Effects of collimated and focused low-intensity pulsed ultrasound stimulation on the mandible repair in rabbits

Introduction

Currently, non-invasive treatment has been a hot topic in the field of orthopedics. Low-intensity pulse ultrasound (LIPUS) is a form of mechanical energy that is transmitted through living tissues as acoustic pressure waves. The United States Food and Drug Administration has approved the use of LIPUS for the treatment of fresh fractures and fracture nonunion (1,2). As a non-invasive treatment, LIPUS has been shown to accelerate fracture healing and repair bone defects by promoting callus formation and stimulating osteogenesis, and its safety and cost-effectiveness have been confirmed (3,4).

Low-intensity collimated pulse ultrasound (LICU) and low-intensity focused-pulse ultrasound (LIFU) are two different forms of LIPUS. LICU is a collimated beam, which is emitted by a transducer into a medium for the treatment. Many studies have shown that LICU can promote the ingrowth, proliferation and early differentiation of osteoblasts in scaffold materials, improving the bone formation (5-7). LIFU is a focused acoustic beam, which has several advantages including irradiation site controllability, strong penetration and low energy attenuation. It has been reported that LIFU can significantly improve the reconstruction of bone defects through enhancing cell proliferation at the defect site (8). LIFU is also effective in the treatment of spinal cord injury (9), neuromodulation (10) and neuromuscular rehabilitation (11).

In this study, the effects of LICU and LIFU on the osteogenesis in the porous silicon carbide (SiC) scaffold implanted in the rabbit mandible were compared. First, temperature at the subcutaneous and subperiosteal tissues was measured during the LICU and LIFU, aiming to confirm the safety of above two treatments. Then, the porous SiC scaffolds were implanted into the rabbit mandible, and the bone ingrowth quantity, bone maturity and bone ingrowth depth under the LICU or LIFU were evaluated at different time points and compared.

Methods

Experimental materials

The porous SiC scaffold was prepared in the Institute of Metal Research, Chinese Academy of Sciences (Shenyang, China) according to the protocol reported previously (12). The characteristics of porous SiC scaffold are as follows: pore size, 800–1,000 µm; porosity, 70–80%; blind hole rate, <1%; compression strength, 30 MPa; elastic modulus, 20 GPa. The materials were sized 10×5×4 mm³, ultrasonically cleaned, autoclaved (134 °C/0.21 MPa) and prepared for use. The crystalline structure of the scaffold was evaluated by X-ray diffraction (XRD, D/max-2500PC, Rigaku) (Figure 1).

Figure 1 XRD image of the porous SiC scaffold. XRD, X-ray diffraction; SiC, silicon carbide.

Experimental animals

Thirty-three adult male Japanese white rabbits (No. SYXK LIAO2008-0005) aged 6 months and weighing 2.8–3.1 kg were used in this study. The rabbits were individually housed in an unrestricted cage and fed ad libitum. These healthy rabbits were allowed to accommodate to the environment for 1 week before experiment. This study was approved by the Animal Ethics Committee of China Medical University.

Subcutaneous and subperiosteal temperatures during LICU and LIFU

Before surgery, six rabbits were intramuscularly anesthetized with 3% pentobarbital at 3 mL/kg (Tianwudr, Tianjin, China). A 1–2 cm incision was made parallel to the mandible, and a thermocouple probe was inserted into the subcutaneous or subperiosteal tissues for the measurement of temperature. The room temperature served as the initial temperature. Ultrasound treatment lasted for 20 min with an ultrasound therapeutic system (Chongqing Haifu Medical Technology Co. Ltd. China), and the temperature was monitored continuously. The parameters of LICU were as follows: frequency, 1.5 MHz; intensity, 30 mW/cm²; pulse width, 200 µs; pulse repetition, 1 kHz. The parameters of LIFU were as follows: frequency, 1.5 MHz; intensity, 300 mW/cm²; pulse width, 200 µs; pulse repetition, 1 kHz; focal spot size, 10 mm × 8 mm; focal distance, 8 mm.

Surgical implantation and LIPUS treatment

Before surgery, 27 rabbits were intramuscularly anesthetized with 3% pentobarbital at 3 mL/kg, and 2% lidocaine (Tianwudr) at 0.3 mL/kg was injected subcutaneously for local anesthesia (13). The surgical sites were parallel to the mandible and the skin, fascia and periosteum were exposed via a 3–4 cm incision using the sterile surgical technique. A rectangular defect (10×5×4 mm³) was created by drilling into the mandibles according to a template. The implant was then gently pressed into the defect, and maintained in close and steady contact with the bone. The animals were intramuscularly injected with penicillin at 1,670 U/mg (Tianwudr) for 3 days after implantation. These animals were randomly divided into three groups: LICU group, LIFU group and control group (sham irritation with the generator power-off). The LICU or LIFU treatment was initiated 24 h after implantation and lasted for 20 min, once daily. The parameters of LICU and LIFU were above mentioned. At each time point (3, 6 and 9 weeks after implantation), three animals were sacrificed and the implant material and approximate 1-cm surrounding bone were collected. The specimens were fixed in 4% paraformaldehyde for 1 week at 4 °C.

Preparation of hard tissue sections

After rinsing with running water for 24 h, the specimens were dehydrated in a series of ethanol solutions (70–100%) and then embedded in methyl methacrylate and dibutyl phthalate solution. The tissue blocks were sectioned longitudinally along the long axis in a diamond-saw microtome (LeicaSP1600, Leica Microsystems, Wetzlar, Germany), and 70–80 µm sections were obtained. Each section was subsequently ground to a thickness of 30–40 µm with the sequential use of #800, #1000, #1200, and #2000 sand papers. There were 3 specimens in each group. The central three sections of each specimen were selected to ensure the comparability among specimens, and five fields were randomly selected from each section for the assessment of bone tissue morphology and quantitative analysis.

Methylene blue-acid fuchsin staining

Sections were stained with methylene blue-acid fuchsin, and the osteogenesis and bone maturity were assessed under a light microscope (OlympusBX 51+DP71, Tokyo, Japan). The bone mass was determined using the image analysis software (Image-Pro Plus 6.0, Media Cybernetics, Rockville, MD, USA). The parameters (brightness, contrast ratio, white balance, exposure time, etc.) were constant during the whole histological examination.

Micro-CT (M-CT) test

Observation of M-CT scanning: The M-CT (Y. Cheetah, Germany) images and data were evaluated using image analysis software (Mimics 16.0, Materialise, Leuven, Belgium). There were 3 specimens in each group. Scanning was performed along the long axis of the implant. A region of interest (ROI) was generated, consisting of bone with material implantation. The M-CT value was chosen to differentiate among materials, host bone and residual pores (including soft tissues) in each specimen, and then reconstruction was performed.

Quantitative analysis of M-CT

(I) Mean pore occupancy fraction (POF): POF was measured as follow: POF = Vbone/(Vbone+Vresidual pore) ×100%, where Vbone is the bone volume and Vresidual pore is the total pore volume (14).

(II) POF in different directions: data were imported to the Mimics software as shown in 2.7.1. Mimics software separated images from a continuous sectional image and generated two masks which were the “bone tissue and material” mask and “20 µm (thickness of M-CT slices) positioning” mask according to the different thresholds. The mask had been generated by 3D reconstruction from different directions (distal to mesial, top to bottom, buccal to lingual) of the material (10×5×4 mm³). The POF of each M-CT slice was measured by Boolean operations (Figure 2).

Figure 2 Schematic diagram of each bone tissue mask and different directions of material. (A) Bone tissue and material mask; (B) 20 µm (thickness of M-CT slices) positioning mask; (C) Boolean operations between two masks (intersects); (D) bone tissue mask; (E) different sections of the material.

Statistical analysis

Data are expressed as the mean ± standard deviation (SD), and one-way ANOVA was used to compare the quantity and structure of bone among groups, followed by Tukey-Kramer test for pairwise comparison. Statistical analysis was done with the SPSS version 21.0 (IBM SPSS Statistics for Windows, Armonk, NY, USA). A value of P less than 0.05 was considered statistically significant.

Results

Subcutaneous and subperiosteal temperatures during LICU and LIFU

For LICU, the subcutaneous and subperiosteal temperatures in all three rabbits varied within 1 °C, and the change in the subcutaneous and subperiosteal temperature was 0.9 °C (Figure 3A) and 0.6 °C (Figure 3B), respectively. For LIFU, the subcutaneous and subperiosteal temperatures in all three rabbits also varied within 1 °C, and the and the change in the subcutaneous and subperiosteal temperature was within 0.9 °C (Figure 3C) and 0.8 °C (Figure 3D), respectively.

Figure 3 Temperature changes during LIPUS. (A) Subcutaneous temperature changes during LICU; (B) subperiosteal temperature changes during LICU; (C) subcutaneous temperature changes during LIFU; (D) subperiosteal temperature changes during LIFU. The temperature variations in both LICU and LIFU groups were no more 1 °C. LIPUS, low-intensity pulse ultrasound; LICU, low-intensity collimated pulse ultrasound; LIFU, low-intensity focused-pulse ultrasound.

Methylene blue–acid fuchsin staining

After methylene blue-acid fuchsin staining, the total bone mass in the LICU and LIFU groups was significantly greater than in the control group, and the mass in the LIFU group was significantly more than in the LICU group at 3, 6 and 9 weeks after implantation (P<0.05) (Figure 4).

Figure 4 Photomicrograph of scaffolds (40×) and analysis of bone area after methylene blue-acid fuchsin staining. (A) LICU group; (B) LIFU group; (C) control group; 3: 3 weeks after implantation; 6: 6 weeks after implantation; 9: 9 weeks after implantation. More bone formation was observed in the LICU and LIFU groups than in the control group at the same time point; a significantly greater area of bone was noted in the LIFU group than in the LICU group. *, P<0.05. LICU, low-intensity collimated pulse ultrasound; LIFU, low-intensity focused-pulse ultrasound.

M-CT

The new bone volume increased over time in the three-dimensionally (3D) reconstructed images of the mesial, distal, and upper areas of the material. The mean POF of the material also increased over time in each group. However, the mean POF in the LIPUS group was significantly greater than in the control group (P<0.05), and that in the LIFU group was markedly greater than in the LICU group at 3, 6 and 9 weeks after implantation (P<0.05) (Figure 5).

Figure 5 Three-dimensionally (3D) reconstructed images from the buccal and lingual surface and the mean POF at 3, 6, and 9 weeks after implantation. (A) LICU group; (B) LIFU group; (C) control group; black: SiC; yellow: new bone. In 3D reconstructed images, the new bone volume in the LIPUS group was greater than in the control group, and the area in the LIFU group was larger than in the LICU group. The mean POF in the LIPUS group was also significantly greater than in the control group, and the POF in the LIFU group was significantly greater than in the LICU group. *, P<0.05. POF, pore occupancy fraction; LICU, low-intensity collimated pulse ultrasound; LIFU, low-intensity focused-pulse ultrasound; SiC, silicon carbide.

Distal-to-mesial

The amount of new bone increased over time in all three groups. At 3, 6 and 9 weeks after implantation, significantly more new bone was observed in both ultrasound-treated groups as compared to the control group (P<0.05); at 3 and 6 weeks after implantation there was no marked difference in the bone mass between the LIPUS groups (3 w: P=0.18; 6 w: P=0.26); at 9 weeks after implantation, significantly more new bone was noted in the LIFU group than in the LICU group (P<0.05).

At 3 weeks after implantation, there was significantly more bone in the mesial and distal areas of the implant than in the middle area in all groups (P<0.05). The curve became less steep at 6 and 9 weeks after implantation; the amount of new bone in the middle of the implant increased, but there was still more bone mass at both ends of the implants than in the middle (Figure 6).

Figure 6 Cartograms of new bone from distal end to mesial end. The distal ends of the scaffolds are indicated as 0 mm, and the mesial ends are indicated as 10 mm. (A) 3 weeks after implantation; (B) 6 weeks after implantation; (C) 9 weeks after implantation; ---, LICU group; …, LIFU group; —, control group; 0 mm →10 mm: distal to mesial.

Top-to-bottom

The amount of new bone increased over time in all three groups. At 3, 6 and 9 weeks after implantation, there was significantly more new bone in the ultrasound-treated groups than in the control group (P<0.05); at 3 and 6 weeks after implantation, there was no marked difference between LIPUS groups (3 w: P=0.21, 6 w: P=0.28); at 9 weeks after implantation, there was significantly more new bone in the LIFU group than in the LICU group (P<0.05).

At 3, 6 and 9 weeks after implantation, there was significantly more bone at the top than in the middle (P<0.05), with the least bone at the bottom of the implant in all groups (Figure 7).

Figure 7 Cartograms of new bone from top to bottom. The top of the scaffolds is indicated as 0 mm, and the bottom is indicated as 5 mm. (A) 3 weeks after implantation; (B) 6 weeks after implantation; (C) 9 weeks after implantation; ---, LICU group; …, LIFU group; —, control group; 0 mm →5 mm: top to bottom.

Buccal-to-lingual

The amount of new bone increased over time in all three groups. At 3, 6 and 9 weeks after implantation, there was significantly more new bone in the ultrasound-treated groups than in the control group (P<0.05), and the amount of new bone in the LIFU group was significantly more than in the LICU group (P<0.05).

In the control group, the new bone in the buccal area of the implant was almost the same to the middle and lingual areas of the implant at 3, 6 and 9 weeks after implantation. The curve was flat in the whole study period. In the buccal area, there was significantly more new bone in the LIPUS group than in the control group at the same time (P<0.05). As depth increased (to lingual), the amount of bone in the LICU group was gradually close to that in the control group, but there was significantly more new bone in the LIFU group than in the LICU group and the control group (P<0.05) (Figure 8).

Figure 8 Cartograms of new bone formation from buccal to lingual. The buccal end of the scaffolds is indicated as 0 mm, and the lingual end is indicated as 4 mm. (A) 3 weeks after implantation; (B) 6 weeks after implantation; (C) 9 weeks after implantation; ---, LICU group; …, LIFU group; —, control group; 0 mm →4 mm: buccal to lingual.

Discussion

LIPUS treatment is an emerging physical therapy for the bone, nerve, and muscle disorders (15). Available studies have confirmed that it can suppress inflammation, promote osteogenesis and facilitate the ossification of hypertrophic chondrocytes (16,17). This study investigated and compared the effects of LICU and LIFU on the bone formation in rabbits. The intensity of LICU was 30 mW/cm² and the intensity of LIFU was 300 mW/cm², both of which are commonly used in clinical practice and studies and have been widely recognized as safe (18-20). In this study, subcutaneous and subperiosteal temperatures were measured continuously during the 20-min LICU or LIFU treatment. Our results showed the subcutaneous and subperiosteal temperatures remained relatively stable and their changes were less than 1 °C. This suggests no damage to cells or tissues (21,22).

As shown in our study, the new bone area in the LIPUS groups was greater than in the control group (P<0.05). Although the mechanism underlying the therapeutic effects of ultrasound is still poorly understood, experiments have shown that LIPUS can promote osteoblast differentiation and bone formation. Cao et al. (7) have confirmed that LIPUS promotes osteoblast differentiation and enhances bone ingrowth and bone formation in porous Ti6Al4V scaffolds. Feng et al. (5) found that both 1 and 3.2 MHz LIPUS promoted the osteoblast differentiation in vitro and enhanced bone maturity in the porous Ti64 scaffolds implanted into rabbit mandibular defects. Our results also confirmed that LIPUS enhances bone formation in the porous SiC scaffolds.

Studies have shown both LICU and LIFU (two different forms of LIPUS) are able to promote osteogenesis. Wu et al. (6) found that LICU facilitated the cellular ingrowth and enhanced the proliferation and early differentiation of osteoblasts in the porous SiC scaffolds. Short-term (2-wk) LICU therapy initiated trabecular bone repair and regeneration in the large trabecular bone defects, whereas cortical bone remained in the initial non-mineralization stage (23). LIFU can also improve the re-ossification through enhancing cell proliferation in the calvarial defect sites (8). However, little is known about the therapeutic effects of LICU and LIFU on the large bone defects. In the present study, results showed the area of bone formation in the LIFU group was greater than in the LICU group, especially in the lingual area of the scaffold (P<0.05). The penetration depth of LIPUS is very important for therapeutic efficacy. The energy of LICU undergoes attenuation to a certain extent as it passes through the skin, subcutaneous tissues, fascia and muscle, and thus whether the ultrasound with required energy can reach the target site is unclear. LIFU has low energy and pulse as in LICU, but has better penetration capability. The intensity of LIFU becomes stronger when it is close to the focal spot, which is sized 10 mm × 8 mm, and thus LIFU is able to encompass the whole scaffold (10×5×4 mm³). The focal distance of LIFU is 8 mm, almost the same as the sum of the depths of the skin (1 mm), subcutaneous tissue (1 mm), muscle (1–2 mm) and scaffold (5 mm). Hence, the energy of LIFU reaching the lingual side of the material is sufficient for the treatment, and more bone formation was observed at lingual side in the LIFU group than in the LICU group. These findings indicate that LIFU accelerates bone formation not only in the buccal area but also in the lingual area of the implant.

Our study indicates ultrasound treatment after implantation of scaffolds is more effective for the large bone defects and provides experimental evidence on the clinical treatment of large bone defects. There were still limitations in this study. Only short-term effectiveness was evaluated, and the optimal parameters of ultrasound and the scaffold should be determined before the clinical application.

Conclusions

Both LIFU and LICU effectively promote bone formation. However new bone formation in the mesial, distal, top, and lingual areas of the implants in the LIFU group are greater than in the LICU group and the control group, especially in the lingual area of the scaffold.

Acknowledgments

We thank Professor Zhang Jingsong at the Institute of Metal Research, Chinese Academy of Sciences for his support with the materials.

Funding: This study was supported by the National Natural Science Foundation of China (NSFC) (No. 81870811) and Natural Science Foundation of the Higher Education Institutions of Liaoning Province (No. LQNK201722).

Footnote

Conflicts of Interest: The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The experiments were approved by the Animal Ethics Committee of China Medical University (No. 2018007).

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