Chinese Medical Journal 2014;127(4):669-674:10.3760/cma.j.issn.0366-6999.20131006
Low-intensity pulsed ultrasound prompts tissue-engineered bone formation after implantation surgery

Wang Juyong, Wang Juqiang, Asou Yoshinori, Paul Fu, Shen Huiliang, Chen Jiani, Sotome Shinichi, Liu Zhao and Shinomiya Kenichi

β-tricalcium phosphate; low-intensity pulsed ultrasound; marrow stromal cells; bone formation
Background A practical problem impeding clinical translation is the limited bone formation seen in artificial bone grafts. Low-pressure/vacuum seeding and dynamic culturing in bioreactors have led to a greater penetration into the scaffolds, enhanced production of bone marrow cells, and improved tissue-engineered bone formation. The goal of this study was to promote more extensive bone formation in the composites of porous ceramics and bone marrow stromal cells (BMSCs).
Methods BMSCs/β-tricalcium phosphate (β-TCP) composites were subcultured for 2 weeks and then subcutaneously implanted into syngeneic rats that were split into a low-intensity pulsed ultrasound (LIPUS) treatment group and a control group. These implants were harvested at 5, 10, 25, and 50 days after implantation. The samples were then biomechanically tested and analyzed for alkaline phosphate (ALP) activity and osteocalcin (OCN) content and were also observed by light microscopy.
Results The levels of ALP activity and OCN content in the composites were significantly higher in the LIPUS group than in the control group. Histomorphometric analysis revealed a greater degree of soft tissue repair, increased blood flow, better angiogenesis, and more extensive bone formation in the LIPUS groups than in the controls. No significant difference in the compressive strength was found between the two groups.
Conclusion LIPUS treatment appears to enhance bone formation and angiogenesis in the BMSCs/β-TCP composites.
Many cell-based approaches have been attempted for skeletal reconstruction in preclinical animal models. For example, bone marrow stromal cells (BMSCs) have been seeded into scaffolds and implanted into animals in many experiments. Although it is easy to obtain bone from porous ceramics, a practical problem impeding clinical translation is the limited bone formation seen in artificial bone grafts.1-3Low-pressure/vacuum seeding and dynamic culturing in bioreactors have led to a greater penetration into the scaffolds, enhanced production of bone marrow cells, and improved tissue-engineered bone formation.2,4,5However, in clinical practice a large scaffold is still often required. Even though the cells may initially be distributed in a uniform pattern, inefficient mass transport will eventually lead to the formation of a necrotic area and cause nonuniformity of the generated tissue.2,6Thein vivostage method study that prompts cell-based tissue-engineered bone formation after implantation is also important.
Ultrasound has been used in numerous fields of medicine, including surgery, diagnostics, and therapeutics. Originally, ultrasound is a source of mechanical energy that causes strain in the tissues that it passes through. Animal and clinical studies have demonstrated that low-intensity pulsed ultrasound (LIPUS) increases the rate of bone formation and the structural strength at fracture sites. LIPUS has also been shown to have a strong positive influence on inflammation, repair, and remodeling of bone fracture.7-10These characteristics suggest that LIPUS may stimulate cell-based bone formation after the implantation surgery and have possible further influences on the biomechanics of engineered bone. To verify this hypothesis, we investigated the effect of LIPUS treatment on bone formation and biomechanical properties of subcutaneous BMSCs/ β-tricalcium phosphate (β-TCP) compositesin vivo.
The total volume of the composite cubes was 125 mm3, with a surface distribution of 5 mm×5 mm×5 mm. The cube was composed of β-TCP2purchased from Olympus Optical Co., Tokyo, Japan. Cube porosity was 75%, with the size of pores ranging from 200 to 400 μm. Pores were interconnected by canals with lengths ranging from 100 to 200 μm.
BMSCs isolation, proliferation, and subculture into porousβ-TCP
Male Fischer rats at 7 weeks of age, with an average weight of 344 g, were used for the donation of bone marrow cells. The methods used to obtain, culture, and subculture bone marrow cells into porous β-TCP have been described previously.2Subcultures were maintained for 2 weeks. The BMSCs/β-TCP composites were then implanted subcutaneously in rats as described below. The experiments were performed at the Tokyo Medical and Dental University and Capital Medical University in accordance with the Care and Use of Laboratory Animals.
Implantation of porous block with BMSCs
After superficial anesthesia by ether inhalation, syngeneic 7-week-old male Fischer rats were anesthetized by intra-abdominal injection of pentobarbital (Nembutal 3.5 mg/100 g body weight). A small incision was made in the middle of the rat’s back, which was then extended subcutaneously by 2.5 cm on either side by forceps. One block of BMSCs was implanted subcutaneously into each side of the back (Figure 1). Wounds were then closed with 4–0 silk sutures.
A total of 96 porous β-TCP blocks and 48 rats were used forin vivoexperiments. Twelve BMSCs/β-TCP composites (six for the ultrasound group and six for the control group) obtained from 12 rats were used for biochemical analysis, and 12 BMSCs/β-TCP composites were used for histological analysis at each time point.
LIPUS treatment
The Sonic Accelerated Fracture Healing System (SAFHS®; Smith & Nephew, Memphis, TN, USA; Teijin Pharma, Tokyo, Japan) generated ultrasound signals consisting of 200 s burst sine waves at 1.5 MHz with an intensity of 30 mW/cm2. These signals were repeated at a frequency of 1.0 kHz. The composite area on the right side of the rat was exposed to LIPUS using a probe (diameter of 25 mm) for 20 minutes each day for a period of either 5, 10, 25, or 50 days (Figure 2).
Biomechanical tests
The composites were harvested for biomechanical tests at 5, 10, 25, and 50 days (12 BMSCs/β-TCP composites at each time point) after implantation. Crosshead speed was performed at 1 mm/min for the compressive strength test in the electronic universal testing machine (EZ Graph, Shimadzu, Japan). Load-deformation curves were obtained from a chart recorder. The compressive strength was determined from the load-deformation curves and the dimensions of each block. Average values were calculated from six measurements. After the biomechanical tests, the composites were collected for alkaline phosphatase (ALP) activity and osteocalcin (OCN) content analysis.
Biochemical analysis
After the biomechanical tests, the ALP activity and OCN (EIA Kit, Biomedical Technologies Inc., Stoughton, MA, USA) content of these composites were measured as described previously.3
Histological and histomorphometric examination
The composites were harvested and used for histological analysis at 5, 10, 25, and 50 days after implantation. For each time point, 12 BMSCs/β-TCP composites (six on the left side and six on the right side) were obtained from six rats for histological examination.
The harvested implants were fixed, decalcified, and embedded. Each block was sectioned with a thickness of 5 mm and stained with hematoxylin and eosin (H&E). For immunohistochemical analysis, deparaffinized sections were treated in turn with 0.4 mg/ml proteinase K (DakoCytomation, Glostrup, Denmark), 3% hydrogen peroxide in methanol, and 5% skimmed milk. They were then incubated with mouse anti-human CD31 monoclonal antibody (1:100 dilution; DakoCytomation) overnight at 4oC. Next, they were allowed to react for 10 minutes with biotinylated pig anti-mouse/rabbit/goat immunoglobulin G (DakoCytomation). These samples were then observed by optical microscopy (IX 70, Olympus, Tokyo, Japan).
Statistical analysis
Average values were expressed as the arithmetic mean±SD. Data analyses were performed using the pairedt-test. Differences were considered to be statistically significant whenP<0.01.
Biomechanical findings
At each time point after implantation, the harvested composites were immediately used for biomechanical test. In both control and LIPUS treatment groups, a similar trend was observed in that the compressive strength increased steeply from 25 to 50 days (Figure 2A). However, no significant difference was found in the compressive strength between the LIPUS groups and control groups (n=6,P>0.05).
Biochemical findings
The ALP activity of implants was measured at 5, 10, 25, and 50 days after LIPUS treatment. ALP activity in the composites of both groups increased steeply toward its peak at 25 days and then decreased to some extent at 50 days. Higher ALP activities, however, were detected in the LIPUS groups at all four time points (Figure 2B). Statistical analysis confirmed that these differences between the LIPUS groups and the control groups were significant at each time point (n=6,P<0.01).
OCN content within the composites was also measured at the same time points. The OCN content in both groups increased positively with time, but likewise the OCN content in the LIPUS treatment group was greater at each time point than the control group (n=6,P<0.01, Figure 2C).
Histological findings
At the designated time point, one incision was made on either side of the rat’s back to expose the BMSCs/β-TCP composites. After 5 days of LIPUS treatment, a large collection of soft tissues was observed closely adhering to the β-TCP composites in LIPUS treatment groups, and the tissues demonstrated a bright red color. Decalcified sections of the LIPUS treatment composites stained with H&E showed a large collection of erythrocytes within the composite pores (Figure 3C). In contrast, the control group had composites that were not surrounded by soft tissues, and the structures surrounding the composites had a dark hue (Figure 3A). Decalcified sections of the control group stained with H&E showed no soft tissues and cells in the composites (Figure 3B).
After 10 days of treatment, the control group demonstrated negligible amounts of vascular endothelial tissues that could be stained with anti-CD31 antibody (Figure 4A). Comparatively, the LIPUS treatment group had a large collection of vascular endothelial cells that could be detected with invasion of fibrous tissues in a large number of the pores (Figure 4B). This was the most obvious characteristic at 10 days of treatment.
At 25 days post-implantation, the LIPUS treatment group demonstrated more primary bone formation in the pores of the composites as compared with the control group (Figure 5A). Bone tissue originated along the surfaces of the pores inβ-TCP, stacked gradually toward the hollow centers, as shown in Figure 5B. Strings of cuboidal cells representing active osteoblasts were observed along the bone-forming surfaces of multiple pore areas, demonstrating more active and progressive bone formation. A significant number of blood vessels could be found within the pores, which supported the progressive bone formation in the LIPUS groups.
By 50 days after LIPUS treatment, the amount of bone formed in the pores had increased in both the control group (Figure 5C) and the LIPUS treatment group (Figure 5D). The greatest quantity of bone tissue originated along the surfaces of the pores in the β-TCP composites. Regenerated bone marrow-like tissues were also observed in association with the newly-formed bone inside some porous regions. These histological changes were more obvious in the LIPUS treatment group than in the control group. In our study, almost all bone formation at the 25- and 50-day time points was fibrous, and no laminar bone formation was detected.
Previous studies have shown that LIPUS treatment can accelerate bone formation, the fracture health by the ultrasound signals consisting of 200 μs burst sine waves at 1.5 MHz with an intensity of 30 mW/cm2is better than other ultrasound intensity, aid in the maturation of calluses for bone fractures, and increase bone stiffness.9-12LIPUS treatment has also been shown to enhance angiogenic, chondrogenic, and osteogenic activities.11,12LIPUS treatment as a therapeutic modality is therefore well established, approved by the US Food and Drug Administration, and is frequently used.12
Transplantation of a porous scaffold seeded with cells is expected to be useful for the treatment of a wide range of patients, including those who have lost large segments of bone due to bone tumors, nonunions, and large congenital defects. A broad range of scaffolds has been proven to be effective at engineering bone tissue, especially when porous scaffolds were used in combination with cells. In clinical practice, however, a large scaffold is often required. Even if uniform cell seeding is initially achieved, inefficient mass transport would result in the eventual formation of a necrotic area, leading to reduction of the generated tissue.6Currently, methods to enhance bone formation in porous scaffolds in the body remain unavailable. To achieve enhanced bone formation, we investigated the effect of LIPUS therapy on BMSCs/β-TCP composites after implantation in our study.
Although numerousin vitroexperiments have been done in regard to applying LIPUS to BMSCs, there are fewin vivostudies that combine LIPUS treatment with the BMSCs/scaffold composites. Hui et al reported that LIPUS treatment of MSCs–calcium phosphate composites enhanced posterior spinal fusion by manual palpation, peripheral quantitative computed tomography, and histomorphometric assessments.13In Hui’s experiment, the transverse processes required decorticate posturing. Thus, bone formation in the composite might not be completely due to the MSCs. In our study, composites were subcutaneously implanted into syngeneic rats, and good bone formation in the LIPUS-treated BMSCs/β-TCP composites was found at each time point after implantation.
ALP and OCN are being used as markers of osteoblast activity.3In our study, ALP activity increased rapidly and peaked at about 25 days, while the changes in OCN content increased steadily over time. This gradual increase in OCN content is well correlated with the course of bone formation in the composites. Histological sections of composites from the LIPUS treatment group exhibited obvious bone formation at 25 days. This bone formation originated along the surfaces of the pores in β-TCP, and could be visualized as stacks growing gradually towards the hollow centers. Strings of cuboidal cells representing active osteoblasts were observed along the bone-forming surfaces of multiple pore areas, demonstrating active and progressive bone formation. Large numbers of osteoblasts was the defining characteristic at 25 days osteocytes appeared in the formed bone. The number of osteoblasts was reduced as compared with Day 25, and this was in accordance with the results of ALP activity and OCN content of the BMSCs/β-TCPcomposites. Using the LIPUS technique, orthopedic surgeons can expect more extensive, reliable, and faster bone formation within the porous scaffold. LIPUS treatment can provide greater benefits in transplant operations, and could therefore increase the advantage of early treatment.
In essence, LIPUS is a type of mechanical energy that is conveyed transcutaneously.10High-frequency, low-magnitude mechanical strain can result in strong regulatory signals to bone tissue.14,15In vitrostudies have shown that LIPUS treatment elevates the mRNA levels of insulin-like growth factor-I, Runx2, OCN, prostaglandin E2, bone matrix protein, ALP, and bone sialoprotein.16-18LIPUS stimulation has also been shown to convert pluripotent mesenchymal cells into the subsequent paths of osteoblasts and chondroblasts.19Furthermore, LIPUS can directly affect osteogenic cells causing mineralized nodule formation.20These previous studies suggest that LIPUS can be a strong initiator of osteogenic differentiation in mesenchymal cells.
In tissue engineering, a biocompatible porous scaffold, into which the cells can be seeded, serves as a template for tissue regeneration. We used porous β-TCP in this study as a bone substitute or scaffold. Due to reluctance in using the scaffold, we wanted to determine if LIPUS treatment prompted bone formation in anin vivostudy. In our experiment, enthusiastic bone formation was seen in the LIPUS treatment group at each time point. It is also possible that increased blood flow and angiogenesis in the LIPUS-treated tissue could play a major role in improving osteogenic differentiation of BMSCs. At 5 days after implantation, a large amount of soft tissues could be visualized closely surrounding the porous β-TCP composite, and these soft tissues were of a bright red color. In the control group, the composites were not surrounded by soft tissues, and the tissues demonstrated a dark hue. Decalcified sections of the control group stained with H&E were significant for the lack of soft tissues and cells in the composites. In contrast, a significant amount of erythrocytes were found in the composite pores of the LIPUS treatment group. LIPUS treatment therefore increased blood flow around the composite site, which suggests that this treatment can stimulate vascularity during wound healing. These data correlate well with the results of Rawoolet al,21who observed a similar vascularity phenomenon in a dog osteotomized ulna fracture model.
Previous studies have shown that ultrasound-mediated microbubble technique, which has been applied in myocardial ischemia and peripheral arterial disease, can promote high expression of VEGF and induce the formation of new blood vessels.22,23Other studies have shown that acoustic cavitation produced by ultrasound results in endothelial cell lysis and angiogenesis without side effects.22,24,25Although the frequency and the intensity of ultrasound used in our study were different from those experiments, a similar histological phenomenon was identified at 5 and 10 days post-LIPUS treatment. Angiogenesis in the early period of LIPUS treatment could be a very important factor in good bone formation of the composites. Angiogenesis in and around the composite site would improve the local blood circulation, and the oxygenic environment could play an essential role in guiding the BMSCs to differentiate into osteoblasts.
Bone formation in both groups at every time point completely consisted of fibrous bone, and no laminar bone was detected in our study. This is a possible explanation as to why no difference in the biomechanical tests could be established between the control and LIPUS treatment groups. Because our research was a short-term study, we did not find increasing biomechanical properties of the BMSCs/β-TCP composites after 50 days of LIPUS treatment, even though the quantity of bone formed was increased. A further long-term investigation of the biomechanical influence of tissue-engineered bone by LIPUS treatment is needed.
In conclusion,LIPUS prompts tissue-engineered bone formation after implantation surgery. The increased bone formation after LIPUS treatment is a result of the direct action in enhancing osteogenic differentiation summed with the indirect action of prompting angiogenesis of the BMSCs/β-TCP composites. LIPUS treatment can therefore be a valuable and convenient technique during the recovery stage of tissue-engineered bone implantation.
Acknowledgments:We thank Olympus Co. Ltd. for kindly donating the β-TCP ceramic blocks. The LIPUS was kindly provided by Teiijin Co. Ltd., Japan.
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(Received April 12, 2013)
Edited by Hao Xiuyuan

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Figure 2. The effect of a LIPUS treatment on the subcutaneously implanted porous blocks with BMSCs for a period of either 5, 10, 25, or 50 days. A: Compressive strength (n=6, P>0.01; P5=0.852, P10=0.722, P25=0.787, P50=0.397). B: ALP activity (n=6, P<0.01; P5=0.006, P10=0.001, P25=0.001, P50=0.001). C: OCN content (n=6, P<0.01; P5=0.001, P10=0.005, P25=0.003, P50=0.005) per block are shown as mean±SD.

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Figure 1. Animal model of the LIPUS treatment. After superficial anesthesia, one composite with BMSCs was implanted subcutaneously into each side of the back. The composite area on the right side of the rat was exposed to LIPUS using a probe for 20 minutes each day from postoperative Day 1.

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Figure 3. The histomorphologic images of the implanted porous composites treated by LIPUS for 5 days. A: Photograph of the composites and their surrounding soft tissues treated by LIPUS for 5 days; left side is for the control group and right side is for the LIPUS group. B: Histological images of the control side. C: the LIPUS side (H&E, Original magnification ×40).

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Figure 4. Representative histological sections of the composites treated by LIPUS for 10 days stained immunohistochemically for the anti-CD31 antibody. A: the control group. B: the LIPUS group (CD31, Original magnification ×100).

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Figure 5.Sections of implants in the control group at 25 (A) and 50 (C) days after implantation, and sections of their counterpart implants in the LIPUS group (B and D). Histological specimens were stained with H&E. Newly-formed bone (arrowhead), osteoblasts (arrow), osteocytes (O), vessels (V), bone marrow-like tissue (M), and β-TCP (T) are indicated (Original magnification ×100).
  1. the National Natural Science Foundation of China (No. 31040029) and the Natural Science Foundation of Beijing, China (No.?3112012).