Previous studies have shown the electromagnetic stimulation improves bone remodeling and bone healing. However, the effect of percutaneous electrical stimulation (ES) was not directly explored. The purpose of this study was to evaluate effect of ES on improvement of bone repair. Twenty-four adult male
Trauma, infection, tumor resection, or skeletal abnormalities can cause bone defects of various shapes and sizes. Many methods have been applied to accelerate bone repair [
Type A gelatin (Bloom number 300, Sigma Chemical Co., Saint Louis, MO, USA) with a mass of approximately 50,000–100,000 Dalton was extracted and purified from porcine skin. A homogeneous 18% gelatin solution was made by dissolving 9 g of gelatin powder in 41 mL of distilled water in a water bath at 70°C. While the gelatin solution was cooling to 50°C, a 20% genipin solution (Challenge Bioproducts Co., Taichung, Taiwan) was added to the gelatin solution to induce cross-linking reactions at a constant temperature. After stirring for 2 min, tricalcium phosphate, Ca3(PO4)2, ceramic particles (Merck, Germany) with grain sizes of 200–300
Twenty-four adult male
Percutaneous electrical stimulation using 2 Hz and 2 mA was applied in this study based on previously successful studies [
shows how the electrodes were positioned. The anode was connected to a point on the back of the neck; the cathode was connected to a front point on the head.
The bone defect regeneration was evaluated radiographically and histologically. Using a micro-CT scanner (SkyScan-1076, Aartselaar, Belgium) and with inhalation anesthesia, each group of animals was examined 4, 8, and 12 weeks after individual percutaneous electrical stimulation. The contrast between the gray levels of the implanted material and the new bone tissue was enhanced. The volume of newly formed bone was evaluated by counting the number of voxels using ImageJ (National Institutes of Health, USA). Next, 3D images of the new bone were obtained using Amira (Visage Imaging GmbH, Berlin, Germany) to evaluate the growth trend.
Anesthetized animals were sacrificed in a carbon-dioxide-filled box 4, 8, and 12 weeks after the operation. The craniectomy sites, along with 2-3 mm of contiguous bone, were removed from each skull after the animal was sacrificed. Specimens were promptly placed into phosphate-buffered 10% formalin and prepared for further analysis. After 24 h of fixation, the specimens were radiographed in a cabinet X-ray machine (MGU 100A, Toshiba Company), using a high contrast X-ray film at 23 keV and 12.5 mA. The craniectomy site radiographs were analyzed using a semiautomatic histomorphometric method, and the regenerated bone was quantitatively evaluated as the percentage of infill area. Using an image analyzer system (Image-Pro Lite, Media Cybernetics, Silver Spring, MD, USA), a satisfactory contrast was achieved between the implanted materials and the new bone tissue by operator selection of a gray level sensitivity standard that was consistent for all treatments. The amount of newly grown bone tissue was calculated by moving a cursor on the digitizing plate, which was visible as a projection over the histological field, and this amount was expressed as a percentage of the ingrowth bone tissue in the created bone defect.
All of the calvarial specimens were subsequently decalcified in a solution of formic acid (10%) for 1-2 weeks and then immersed in sodium sulfate overnight. The specimens were dehydrated in a graded series of ethanol and then embedded in a tissue freezing medium (OCT). Axial sections of the decalcified bone and implants (10
All numerical data were presented as the mean ± one standard deviation. Significant differences among the samples were evaluated using Student’s
All animals in both the experimental and control groups survived for the entire experimental period without any local or general complications. There was no wound infection, scalp effusion, hematoma, festers, or disturbed wound healing at the surgical site of the calvarial bone. The results reveal that the GGT composite did not lead to histopathology or exhibit poor biocompatibility with the peripheral osseous tissues. No gaps between the GGT composite and the peripheral osseous tissues were noted, and no GGT composite was extruded (Figure
This figure represents the gross examination. (a) illustrates that there was no wound infection, scalp effusion, hematoma, festers or disturbed wound healing at the surgical site of the calvarial bone. No gaps between the GGT composite and the peripheral osseous tissues were noted, and no GGT composite was extruded. (b) shows that the brain tissues underlying the implantation site did not display any evidence of cortical inflammation, scar formation, or necrosis.
Gross examination does not determine whether the newly formed osseous tissues were completely calcified new bones. Therefore, X-ray radiographs were obtained for further analysis. The performance both with and without ES in repairing the calvarial bone defect was evaluated to determine the efficacy of ES in accelerating the healing of defective bones. Figure
This figure shows the X-ray images. The top of each image corresponds to the front part of the rats, where the cathode is connected, and the bottom is the back part, where the anode is attached. (a), (c), and (e) display the results of the ES groups after 4, 8, and 12 weeks of bone repair, respectively. (b), (d), and (f) show the corresponding images of the control groups, in which non-ES was performed. In the ES groups, the new bone mostly formed on bilateral sides, whereas the new bone was U-shape in the non-ES groups (NB: new bone; GGT: genipin cross-linked gelatin mixed with tricalcium phosphate).
The new bone formation became more obvious as the time between implantation and examination increased. Additionally, the radiographs clearly reveal that the calvarial bone defect was repaired gradually and that the GGT composite degraded progressively. Figure
This figure shows that the percentage of bone regeneration in the rats is significantly higher in the ES groups than in the non-ES groups. The bone regenerated appreciably from 4 to 8 weeks and from 8 to 12 weeks.
A new three-dimensional (3D) method, micro-CT, was applied to evaluate the amount of new bone formation. Bone repair in both the ES and non-ES groups were evaluated. Figure
This figure shows the 3D images of the new bone. The top of each image corresponds to the front part of the rats, where the cathode is connected, and the bottom is the back part, where the anode is attached. (a), (c), and (e) display the results of the ES groups after 4, 8, and 12 weeks of bone repair, respectively. (b), (d), and (f) are the corresponding images of the control groups, in which non-ES was performed. In the ES groups, the new bone mostly formed on bilateral sides, whereas the new bone was U-shape in the non-ES groups.
In the non-ES groups, four weeks after surgery, some newly formed bone evidently grew into the GGT construct (Figure
The images obtained four weeks after surgery displayed a larger amount of new bone formation in the ES groups than in the non-ES groups (Figure
Table
The volume of new bone formation measured with micro-CT scan.
Implantation time | With/without ES | Volume (mm3) |
---|---|---|
Four weeks* | With ES | 16.70 ± 2.62 |
Without ES | 12.36 ± 2.24 | |
Eight weeks* | With ES | 22.03 ± 2.84 |
Without ES | 16.31 ± 2.49 | |
Twelve weeks** | With ES | 27.85 ± 2.16 |
Without ES | 18.29 ± 1.57 |
The volume of the newly-formed bone was determined in each implantation period (
A histological evaluation was performed to compare the progress of restoration at the bone defect in the ES and non-ES groups. Figure
This figure shows transverse histological sections of calvarial defect 12 weeks after implantation with H&E stain. (a) displays the result of bone repair in the ES group. (b) shows the corresponding event for the control groups, in which non-ES was performed. The amount of bone regeneration in the rats is higher in the ES group than in the non-ES group (ITF: interface; NB: new bone; HB: host bone; FBC: foreign body capsule; original magnification: 40).
Longitudinal sections with the ALP and TRAP stains identified the activity of osteoblasts and osteoclasts near the electrodes. In Figures
This figure shows longitudinal sections with ALP stain. The left side represents the far-end, in the direction of the cathode; the right side indicates the near-end, near the anode. For the ES group, the ALP accumulates on the left side after 12 weeks, as shown in (a); (b) illustrates the uniform distribution of ALP in the non-ES groups. The results indicate that osteoblasts are more active near the cathode in the ES groups.
This figure shows longitudinal sections with TRAP stain. The left side represents the far-end, in the direction of the cathode; the right side indicates the near-end, near the anode. (a) shows the aggregation of TRAP on the right side in the ES group after 12 weeks. (b) illustrates the uniform distribution of TRAP in the non-ES groups. The results demonstrate that osteoclasts are vigorous near the anode in the ES groups.
The GGT composite did not cause an obvious cytotoxic reaction in rabbits [
As shown with the data in Figure
The present study only investigated the appearance of bone regeneration using X-ray and micro-CT imaging. The hypothesis that the percutaneous electrical stimulation can accelerate the bone regeneration was confirmed in this study. However, the mechanism is still not very clear. The further study focused on the in-depth mechanism of ES which is undergoing.
The study used a calvarial bone defect model to evaluate the effect of percutaneous electrical stimulation on bone regeneration. Radiographic analyses including X-ray and micro-CT show progressive bone healing with time. The bone repair rate is higher in the ES groups than in the non-ES groups. Additionally, the new bone grows on bilateral sides in the ES groups and accumulates in a U-shape in the non-ES groups. Histological evaluations with H&E stain also confirm the higher new bone formation rate in the ES groups. The slides with ALP and TRAP stains indicate that osteoblasts are more active near the cathode and that osteoclasts are more vigorous beside the anode. The results prove the thesis that percutaneous electrical stimulation can accelerate bone repair, and the bone regeneration is more active near the cathode than around the anode. Bone repair might rely on the activity of osteoblasts and osteoclasts by EA stimulation.
All authors state that they have no conflict of interests.
B.-Y. Yang and T.-C. Huang contributed equally to this work. Y.-S. Chen and Chun-Hsu Yao contributed equally to this work.
The present research is financially supported by the National Science Council of the Republic of China, Taiwan (contract no. NSC 98-2221-E-039-005-MY3) and the China Medical University (contract no. CMU99-S-44). The authors do not have a direct financial relation with the commercial identity mentioned in the paper that might lead to a conflict of interest for any of the authors.