不同剂量微波治疗对骨折术后钛合金植入物毗邻组织的影响

doi:10.3969/j.issn.2095-4344.0119

中国组织工程研究 ›› 2018, Vol. 22 ›› Issue (7): 1072-1077.doi: 10.3969/j.issn.2095-4344.0119

• 骨科植入物 orthopedic implant • 上一篇下一篇

不同剂量微波治疗对骨折术后钛合金植入物毗邻组织的影响

王刚，徐义明，叶冬梅，付腾飞，邹玉珍，冯宪煊，白跃宏

上海交通大学附属第六人民医院康复医学科，上海市 200233

出版日期:2018-03-08 发布日期:2018-03-08
通讯作者: 白跃宏，博士，教授，博士生导师，上海交通大学附属第六人民医院，上海市 200023
作者简介:王刚，男，1982年生，河南省新郑市人，汉族，2016年上海交通大学毕业，博士，主治医师，研究方向是骨关节疾病康复的基础与临床研究。
基金资助:
上海市科学技术委员会科研计划项目(13231202600)

Effects of different doses of microwave therapy on adjacent tissue of titanium alloy implants after fracture surgery

Wang Gang, Xu Yi-ming, Ye Dong-mei, Fu Teng-fei, Zou Yu-zhen, Feng Xian-xuan, Bai Yue-hong

Department of Rehabilitation, Affiliated Sixth People’s Hospital of Shanghai Jiao Tong University, Shanghai 200233, China

Online:2018-03-08 Published:2018-03-08
Contact: Bai Yue-hong, M.D., Professor, Doctoral supervisor, Department of Rehabilitation, Affiliated Sixth People’s Hospital of Shanghai Jiao Tong University, Shanghai 200233, China
About author:Wang Gang, M.D., Attending physician, Department of Rehabilitation, Affiliated Sixth People’s Hospital of Shanghai Jiao Tong University, Shanghai 200233, China
Supported by:
the Scientific Research Program of Science and Technology Commission of Shanghai City, No. 13231202600

摘要/Abstract

摘要：

文章快速阅读：

文题释义：

微波治疗：作为一种常用的高频电疗，在骨关节疾病临床康复治疗中有重要作用，能够均匀作用于皮肤和深部组织，促进治疗处血液循环，显著增加局部血液流量，为需要恢复的损伤组织提供更多的营养物质同时带走局部代谢废物及坏死组织。还具有镇痛作用，对神经性疼痛、肌肉痉挛引起的疼痛、因肿胀发生的张力性疼痛和炎性疼痛均有非常好的止痛效果。

钛合金植入物：与传统的不锈钢植入物相比，钛合金植入物密度小、强度大，生物相容性更好，弹性模量与骨更接近，是目前最适合人体应用的骨折手术植入物材料。并且在物理特征方面，钛合金的导磁性和导电性低，在体外也发现其界面电磁加热效应小。相对于不锈钢等金属植入物，微波治疗可能更适合应用于钛合金植入物固定后患者，钛合金植入物毗邻的组织受到的影响会更小。

摘要

背景：微波治疗是临床常用的康复物理治疗方法，具有温热效应，能够促进治疗部位血液循环，可被用来促进损伤的骨组织恢复。通常，金属植入物被列为微波治疗的禁忌证。

目的：观察微波治疗下钛合金植入物周围组织的热损伤情况。

方法：将44只新西兰白兔随机分为植入物组和对照组。植入物组构建新西兰白兔股骨中段骨折动物模型，对骨折模型处给予钛合金内固定系统固定。对照组仅造模。2组在术后3 d开始对白兔股骨中段骨折处行连续30 d的2 450 MHz微波治疗，剂量为20 W或40 W，每天治疗20 min。

结果与结论：植入物组和对照组经20 W的微波干预后，植入物毗邻组织温度没有明显上升，且没有出现严重组织热损伤；而经40 W的微波干预后，植入物毗邻组织温度明显上升，且40 W植入物组植入物毗邻组织损伤明显。结果提示低剂量的微波治疗可用于治疗骨折钛合金植入物固定术后的康复治疗。

中国组织工程研究杂志出版内容重点：人工关节；骨植入物；脊柱；骨折；内固定；数字化骨科；组织工程
orcid: 0000-0001-6387-6382(Bai Yue-hong)

关键词: 微波, 钛合金, 线粒体, 骨科植入物, 透射电子显微镜, 肌肉组织, 神经组织, 植入物, 动物实验

Abstract:

BACKGROUND: Microwave treatment is a common physical therapy method that can increase the temperature and blood circulation of deep tissues, and is used for improving fracture repair. However, microwave treatment cannot be used if there is surgically implanted metal plate or screw.

OBJECTIVE: To observe the dame of microwave treatment to the tissues surrounding the titanium alloy implants.

METHODS: Forty-four New Zealand white rabbits were randomized into experimental and control groups. The model of the fracture at the middle of the femur was established in all rabbits, and the rabbits in the experimental group were implanted with titanium alloy internal fixation systems. A 30-day microwave treatment (2 450 MHz, 20 W or 40 W, 20 minutes daily) was applied to the fracture site in all rabbits at 3 days after operation.

RESULTS AND CONCLUSION: After 20 W of wave microwave treatment, the temperature of tissues around the implants showed no significant increase or severe heat injury. While, 40 W of wave microwave treatment significantly increased the temperature of tissues around the implants and the tissue was damaged severely. Our results indicate that, the low-dosage microwave treatment may be a promising method in the rehabilitation therapy of fractures with titanium alloy internal fixation.

中国组织工程研究杂志出版内容重点：人工关节；骨植入物；脊柱；骨折；内固定；数字化骨科；组织工程

Key words: Microwaves, Prostheses and Implants, Titanium, Tissue Engineering

中图分类号:

R318

王刚，徐义明，叶冬梅，付腾飞，邹玉珍，冯宪煊，白跃宏. 不同剂量微波治疗对骨折术后钛合金植入物毗邻组织的影响[J]. 中国组织工程研究, 2018, 22(7): 1072-1077.

Wang Gang, Xu Yi-ming, Ye Dong-mei, Fu Teng-fei, Zou Yu-zhen, Feng Xian-xuan, Bai Yue-hong. Effects of different doses of microwave therapy on adjacent tissue of titanium alloy implants after fracture surgery[J]. Chinese Journal of Tissue Engineering Research, 2018, 22(7): 1072-1077.

图/表（结果） 3

Quantitative analysis of animals

None rabbits in the study dropped out, and all were involved in the result analysis.

Temperature changes

During microwave irradiation, we simultaneously measured the temperatures in deep muscle tissues. Temperature changes are shown in Figure 2. It was found that the temperature in the implanted group at 40 W was higher than that in the implanted group at 20 W (P < 0.000 1). In addition, the temperature in the implanted group at 40 W was higher than that in the control group at 40 W (P = 0.037 3). However, there was no significant difference between control and the implanted groups at 20 W (P = 0.547 9). The peak temperature of the deep muscles in the implanted group at 20 W was between 37.5 ℃ and 37.7 ℃. The peak temperature of the deep muscles in the implanted group at 40 W was between 40.3 ℃ and 40.6 ℃.

Light microscopy

Sections of the thigh muscle in each group were stained with hematoxylin and eosin after 30 days of microwave treatment. No abnormal morphological changes were found either in the control or implanted groups. Sections of sciatic nerve from each group were also stained with hematoxylin and eosin after the 30-day microwave treatment. The myelin sheaths of the sciatic nerves in the implanted group at 40 W were found to be swollen (Figure 3).

Transmission electron microscopy

Transmission electron microscope examinations of the skeletal muscle and sciatic nerves in each group were conducted after the 30-day microwave treatment. The muscles in the implanted group at 20 and 40 W showed mitochondrial swelling and vacuolization (mitochondrial damage). Myofilaments and myocommata were unclear. The myelin sheath of the sciatic nerves adjacent to the fractures at 20 W was normal. Part of the myelin sheath of myelinated nerve fibers showed lamellar separation, and ball-like changes in the pith were also observed. The mitochondria in the axons of the implanted group at 40 W were normal (Figure 4). No abnormal morphological changes in the sciatic nerve were discovered in the control group or the implanted group at 20 W.

A KS400 image analysis system (Zeiss) was applied to calculate the percentage of the damaged mitochondrial area in each sample, to allow the quantitative analysis of mitochondrial damage in the muscle between two implanted groups, and found significant differences (Figure 5). The percentage of the damaged myelin sheaths of sciatic nerves adjacent to the fractures in the implanted group at 40 W was (31% ± 5%), while the myelin sheaths of sciatic nerves adjacent to the fractures in the implanted group at 20 W were normal. Thus, muscle and nerve injuries in the implanted group at 40 W were more serious than those at 20 W.

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引言

Orthopedic physicians have considered the internal fixation system to be the preferred method for the treatment of fractures that need fixation surgery^[1]. However, some patients suffer from symptoms after surgery, such as pain associated with the implants, delayed union, or non-union. Microwave therapy, an ordinary type of physical therapy method, can raise the temperature of deep tissues to 41 ℃ -45 ℃, alleviate pain^[2], increase the blood flow up to 15 times^[3-4], and change the physical properties of fibrous tissues^[5-6]. Thus, microwave therapy is applied in the treatment and rehabilitation of muscle, tendon, and bone injuries^[6-10]. However, contraindications for microwave therapy that have been extensively documented in the literature include surgically implanted metal plates, screws, or pins in the treatment field. As a type of electromagnetic wave, microwave radiation can be reflected, refracted, or border spread^[7]. In addition, the eddy current that is stimulated by the electromagnetic field may cause Joule heating of the implants. All of these factors will lead to a sharp rise in temperature and even heat damage in local tissues^[11-12]. Previous in vitro studies have found that the temperature of the implanted metal plate may cause tissue ambustion in the frequency fields of 900 MHz and 27 MHz^[13]. However, some doctors believe that this contraindication seems to have arisen from “common sense”, rather than evidence-based practice. In vitro studies of radio frequency (RF) electromagnetic fields suggested that the presence of the implant causes little risk, even though 1 800 MHz and 2 450 MHz ^[15-16]microwave radiation increased the specific absorption rate (SAR) values. Furthermore, Seiger and Draper ^[17]increased the range of motion of the ankle joint of the patients with titanium metal implants by pulsed shortwave diathermy, and no discomfort, pain, or burning sensations are reported. Compared with medical stainless steel, titanium or titanium alloy has better biocompatibility and its elasticity is closer to that of bone tissue^[18]. Titanium alloy is a type of medical implant with low magnetic permeability and electric conductivity, and lower RF heating was observed at the border of the titanium alloy implants and phantom in vitro^[19-20]. However, few morphological studies have been published concerning the effects of titanium alloy internal fixations on nearby muscles and nerves in microwave treatments. The relationships between titanium alloy internal fixations, morphological changes, and microwave doses still remain unknown.

A previous study found that titanium alloy implants did not increase temperatures significantly in animals using 25 W microwave treatment and that microwave treatment promoted fracture healing^[21]. To study the

dosage of microwave treatments that should be applied to the healing of fractures with titanium alloy internal fixations, we investigated the morphological changes of muscles and sciatic nerves near the fixations using different dosages of microwave treatment for a 3.0 mm transverse osteotomy in the middle of the femur, and discussed the reasons for the morphological changes observed.

中国组织工程研究杂志出版内容重点：人工关节；骨植入物；脊柱；骨折；内固定；数字化骨科；组织工程

材料方法

Design

To check the types of morphological changes that microwave treatment in healing tissues with fractures with titanium alloy internal fixation.

Time and setting

The trial was finished at the Animal Laboratory Center and Department of Rehabilitation of Shanghai Sixth People’s Hospital from February to July 2015.

Materials

Forty-four male healthy adult New Zealand white rabbits (12-18 months) from the Laboratory Animal Center (Approval number: SYXK (Hu) 2011-0128), weighing 2.0-3.2 kg were used in this study. The animals were kept at the Animal Laboratory Center at Shanghai Sixth People’s Hospital, and were housed in a temperature (25℃), humidity (7%), light (light on from 8 a.m. to 8 p.m.) and diet (food at 8 a.m., water at 4 p.m.)-controlled environment.

The internal fixation system (LCP, T-plate 2.4) which was composed of a titanium alloy plate (4.60 ± 0.24 × 0.42 ± 0.08 cm) and screws was provided by Synthes Company, USA.

Methods

Grouping

Forty-four New Zealand white rabbits were randomized into experimental and control groups. The model of the fracture at the middle of the femur was established in all rabbits and the rabbits in the experimental group underwent implantation of titanium alloy internal fixation systems. A 30-day microwave treatment (2 450 MHz, 20 W or 40 W, 20 minutes daily) was applied to the fracture site in all rabbits at 3 days after operation.

Animal model construction

Rabbits were anesthetized with an intravenous injection of pentobarbital sodium (30 mg/kg; Shanghai Xinya Pharmaceutical Co., Ltd. 2006006). The femur was exposed via a longitudinal skin incision on the outside of the right hind limb. To reduce pain during the postoperative and recovery periods, a transverse osteotomy with a 3.0 mm bone defect instead of a femur complete transverse fracture was created in the middle of the femur. The internal fixation system was implanted into the right femoral upper end of the experimental group (Figure 1). To prevent infection and reduce postoperative pain, all rabbits were injected intramuscularly with penicillin (Shanghai Xianfeng Pharmaceutical Co., Ltd. Chinese medicine quasi word H31020930) (800 000 U) and a surgical dressing was applied to the incision once daily during the 3 days following surgery. Afterwards, the rabbits were killed by air embolism.

Microwave treatment

Three days after operation, microwave therapy was applied to all rabbits. The treatment regimen involved the right upper thigh. The applicator (RM-170A, ITO Company, Japan) was connected to a 2 450 MHz microwave generator (PM-800, ITO Company, Japan) with power output ranging from 0 to 200 W. The antenna was in the applicator (RM-170A, ITO Company, Japan), and the diameter of the applicator was 13.3 cm. The 20 and 40 W continuous-wave microwave exposures lasted for 20 minutes per day. The non-contact applicator was perpendicular to the lesion and 10 cm away from the skin. To exclude the influence of the changes of body temperature and other factors over the course of the day, we provided all microwave treatment at the same time.

Temperature measurements

Three rabbits from each group were used in this experiment. We used an anti-interference couple thermometer (FHC, ME-04008, BOWDOINHAM, USA) for the temperature measurements. After the rabbits were anesthetized with an intravenous injection of pentobarbital sodium (30 mg/kg), we placed 8 cm of thermal probes into the deep muscles, which were 5 mm above the middle hole of the implanted titanium alloy plate. We separated the biceps femoris and the vastus lateralis muscles by blunt dissection to place the thermal probes in the muscle, placing the probes with the recording side facing the biceps femoris. The temperature was recorded every minute for 20 minutes when the 20 and 40 W microwave treatments were applied. The temperature of the laboratory was maintained at 24 ℃.

Light microscopy

Eight rabbits from each group were sacrificed under anesthesia using an intravenous injection of pentobarbital sodium (30 mg/kg), and eight samples of muscular and nervous tissues around the implants or fractures without implants were obtained. The specimens were fixed in 10% formalin. After being embedded in paraffin, 5 μm-thick longitudinal sections were prepared and stained with hematoxylin and eosin following standard techniques for microscopic examination (Chongqing Photoelectric Instrument Factory B200i).

Transmission electron microscopy

Eight rabbits from each group were sacrificed under anesthesia with an intravenous injection of pentobarbital sodium (30 mg/kg). Eight samples of muscular and nervous tissues around implants or fractures without implants were fixed with 2.5% glutaraldehyde (Shandong Chemical Co., Ltd. 20140604) for 16 hours at 4 ℃, post fixed with 2% OsO₄ for 2 hours at 4 ℃, dehydrated by an ascending ethanol series, passed through propylene oxide, and then embedded in resin. Ultra-thin sections (90 nm in thickness) on mesh grids were stained with uranyl acetate and lead acetate, and examined with an electron microscope.

To carry out quantitative analysis of mitochondrial damage of muscle in the two implanted groups, we calculated the percentage of damaged mitochondrial area in each sample using the KS400 image analysis system (Zeiss). To perform quantitative analysis of myelin sheath damage to sciatic nerves in the implanted group at 40 W, we calculated the percentage of damaged myelin sheath by scoring 100 myelin sheaths under transmission electron microscope.

Main outcome measures

Temperature changes in the muscle tissues around the implants; the morphological changes of the muscle tissues around the implants under light microscopy and transmission electron microscope.

Statistical analysis

Statistical analyses of the temperatures were performed with SAS 9.1 (SAS, North Carolina, USA), and the other analyses were performed with SPSS 19.0 (SPSS, Chicago, IL, USA). All data are presented as the mean ± SD. Differences between groups were tested using t-test. P < 0.05 was considered statistically significant.

中国组织工程研究杂志出版内容重点：人工关节；骨植入物；脊柱；骨折；内固定；数字化骨科；组织工程

讨论

In general, metallic implants in the treated regions are described as a contraindication for RF treatment because of intense and high heating. As an alternative, microwaves were analyzed in this study to evaluate the influence of titanium alloy internal fixations in microwave treatments for fractures and discuss the damage threshold. The main findings of this study were that titanium alloy internal fixations did not markedly increase the temperature of tissues using continuous-wave microwave treatment at 2 450 MHz, 20 W, for 20 minutes per day compared with the control group. However, the temperature was remarkably increased in the implanted group at 40 W. Titanium alloy internal fixations induced morphological changes in the skeletal muscles in the implanted groups, and in the sciatic nerves in the implanted group at 40 W in the microwave treatment of fractures. The discussion focuses on these findings, alongside the possible mechanisms of the morphological changes observed in the muscles and nerves around the titanium alloy implants in 20 and 40 W microwave treatments.

Microwave is an important physical therapy technique in clinical treatment, and its main therapeutic effect is the raising of local tissue temperature to allow more efficient relief of pain, stimulate repair processes, reduce muscle and joint stiffness, and increase tendon extensibility^[2]. Our results indicate that titanium alloy internal fixation increased temperatures in the healing of fractures using 40 W microwave treatment, compared with the non-implanted control group and the implanted group at 20 W. Using the Repeated Measures Analysis of Variance of SAS 9.1, the temperatures following 20 minutes of treatment with the implanted group at 40 W were significantly higher than those from the implanted group at 20 W. Moreover, the temperatures for 20 minutes of treatment with the implanted group at 40 W were also significantly higher than those from the non-implanted control group at 40 W. Although the temperatures for 20 minutes of treatment showed no significant difference between the non-implanted control group and the implanted group at 20 W, the temperatures of the implanted group at 20 W were higher than those of the non-implanted control group at most time points. Generally speaking, if temperatures are higher, more thermal damage occurs to the muscles and nerves. However, the damage was not solely related to temperature because of the metal implants used in our study. The temperatures of the non-implanted control groups at 40 W were higher than those of the implanted group at 20 W (P=0.000 2), and there were no abnormal morphological changes in the non-implanted control groups at 40 W.

In our study, thigh muscles from the non-implanted control groups and the implanted groups stained with hematoxylin and eosin after the 30-day microwave treatment had no abnormal morphological changes at different microwave treatments. Moreover, TEM of skeletal muscles from the implanted groups showed mitochondrial swelling and vacuolization. The reason why myofilaments and myocommata were unclear was that elevated temperature accelerated blood circulation and tissue fluid exudation into muscle cells was increased. Previous studies have found that microwaves can cause changes in mitochondria^[22-23]. We used TEM to analyze the fine details of changes in cellular organelles, and found muscular mitochondrial swelling and vacuolar changes. In addition to thermal effects, these changes may be related to the non-thermal effects of microwaves^[24-25]. Mitochondria play an integral role in apoptotic cell death. It is widely known that mitochondrial swelling is one of the most important indicators of the opening of the mitochondrial permeability transition pore. The opening of channels or swelling sufficient to rupture the outer mitochondrial membrane may cause the release of cytochrome c, which, in turn, leads to necrotic or apoptotic cell death^[26]. Because of titanium alloy internal fixations, the mitochondria of the muscles near the implants swelled, whereas those from the non-implanted control groups and the axons of the implanted group at 40 W were normal. The reason maybe that the muscles near the metal implants received a higher dose of RF irradiation with the reflection of electromagnetic waves from the metal surfaces. The implant may couple with the RF source, thus inducing a current on the implant surface. The induced current further produces a secondary EM ?eld and so the implant acts as a weak radiating antenna in tissues^[14]. As a kind of electromagnetic wave, microwave irradiation can be reflected, refracted or transmitted at boundaries^[7]. Two types of effects can be ascribed to microwaves, i.e. thermal and non-thermal. The changes of muscular mitochondrial may be related to the non-thermal effects of microwaves. More microwaves damaged more mitochondria in the implanted groups at 20 W and 40 W, and mitochondrial injuries were more serious at 40 W.

We also found that TEM of sciatic nerves in the implanted group at 40 W showed myelin sheath swelling and lamellar separation of part of the myelin sheath of myelinated nerve fibers, forming a ball-like pith change that was similar to demyelination. The myelin sheath of the sciatic nerve in the implanted group at 40 W stained with hematoxylin and eosin after 30-day microwave treatment also swelled. The myelin sheath injuries were serious at 40 W. Myelin swelling can be observed in obstructive jaundice, cerebral ischemia, poisoning, and diabetes^[27-31], and is also related to hyperthermia. Hoogaveen et al.^[32] found that morphological changes in sciatic nerves in rats were related to the treatment temperatures. Treatment at 44 ℃ led to edema formation within 24 hours and segmental demyelination within 1 week after treatment. Edema, severe damage to endothelial cells, and vascular obstruction were the most remarkable changes 2 and 4 hours after 45 ℃ treatment. Similar histopathological results were observed in larger animal models after 60 minutes of intraoperative radiation therapy treatment using temperatures of 43 ℃, 44 ℃, or 45 ℃^[33]. TEM of the sciatic nerve in the implanted group at 40 W did not reveal mitochondrial swelling or vacuolar changes. This was different from the changes in the muscles of the implanted groups. Why the muscle mitochondria in the implanted groups at 20 W damaged, while the mitochondria of the nerves were not? We think that there may be two main reasons. First, the muscles near the metal implants received a higher dose of RF irradiation from the reflection of electromagnetic waves from metal surfaces. Second, muscle tissues with high water content absorb more energy.

Microwave hyperthermia is a physical therapy technique that raises the temperature of a given tissue. The threshold required for the induction of thermal damage to muscle in pig has been previously reported to be 46.0 ℃ over 30 minutes^[34]. This is higher than the temperature and time in our study. Titanium alloy internal fixations caused much less damage to the muscles and nerves using 20 W microwave treatment for fractures, with peak temperatures between 37.5 ℃ and 37.7 ℃. Previous studies have found that the mitochondria could change size in a phasic manner, swelling when cells were exposed to a temperature of 40 ℃, and that all of these shape changes were reversible when the temperature returned to normal ^[35-36]. Thus, 20 W microwave treatment may be a promising method in the healing of fractures with titanium alloy internal fixation.

In summary, our in vivo study proved that titanium alloy internal fixations dose not dramatically increase the temperature in muscle tissues with continuous-wave microwave treatment, at 20 W. Titanium alloy internal fixations cause morphological changes to the muscles and nerves. The morphological changes are related to the dosage of the microwave treatment, and the 20 W microwave treatment caused less morphological changes to the muscles and nerves near the fixations compared with the 40 W microwave treatment. Our results indicate that in the healing of fractures with titanium alloy internal fixation, a low dosage of microwave treatment makes less damage to muscles and nerves, and it may therefore be a promising method.

中国组织工程研究杂志出版内容重点：人工关节；骨植入物；脊柱；骨折；内固定；数字化骨科；组织工程

不同剂量微波治疗对骨折术后钛合金植入物毗邻组织的影响

Effects of different doses of microwave therapy on adjacent tissue of titanium alloy implants after fracture surgery

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