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We aimed to establish a novel approach with 3D high frequency power Doppler ultrasonography (3D-HF-PDU) to assess microvasculature at the fracture site in rat femurs by comparing with microCT-based microangiography. Twenty-four 9-month-old ovariectomized (OVX) osteoporotic rats and age-matched sham-ovariectomized (Sham) rats were used for establishing closed fracture models on right femora. At 2, 4, and 8 weeks post-operatively, four rats in each group underwent in vivo 3D-HF-PDU scanning for evaluation of vascularization and blood flow at the fracture site. Then the fractured femora were harvested for ex vivo microangiography, and neovasculatures within the callus were reconstructed for vascular volume analysis. Correlation between the vascular volumes of the two methodologies was examined. Both 3D-HF-PDU and microangiography showed a decline of vascular volume at the fracture site from 2 to 8 weeks and a significantly larger volume in the Sham group than the OVX group. A significant linear positive correlation (r = 0.87, p < 0.001) was detected between the volumes measured by the two methodologies. Osteoporotic rats had a diminished angiogenic response and lower blood perfusion than Shams. We believe 3D-HF-PDU is feasible and reproducible for in vivo assessment of microvasculature during femoral fracture healing in rats. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 30:137–143, 2012
Angiogenesis plays a critical role in fracture healing especially in osteoporotic bone,1, 2 but the mechanisms that govern angiogenesis and their influences on impairing blood supply during the osteoporotic healing process remain poorly understood. The rat fracture model is widely used in fracture healing studies.3–5 However, quantitative techniques for studying vascularization, such as microCT-based microangiography, require animals to be euthanized and evaluated ex vivo.6–8 An in vivo, yet repeatable approach would allow quantitative measurements to serially monitor the revascularization at the fracture site and to study differences in microcirculation between fracture healing in normal and osteoporotic rats.
Power Doppler ultrasonography is based on the integrated Doppler power spectrum, which can be used to assess moving objects. In the vascular system, the power of the Doppler signals in relation to the number of moving erythrocytes can be detected and displayed in a color scale.9, 10 Due to its non-invasiveness and high sensitivity to slow flow, power Doppler in 2D was used to detect neovascularization in vivo with fracture healing.11–13 By using 3D power Doppler ultrasonography, reconstructed 3D vascular trees within a volume of interest (VOI) can be studied clinically. The vascularization can be objectively evaluated using specialized imaging software.14–17 However, due to the relatively narrow dynamic range of flow detection, the most frequently used clinical ultrasound frequency is unsuitable for studying slow flow in small animals. To improve detection capability and sensitivity, high frequency ultrasound is recommended for studying microvasculature in small animal models.18
To understand revascularization and local blood perfusion during normal and osteoporotic fracture healing, 3D high frequency power Doppler ultrasonography (3D-HF-PDU) was utilized and compared with microCT-based microangiography. Our aim was to validate the application of 3D-HF-PDU imaging for in vivo qualitative and quantitative assessment of microcirculation and angiogenesis in a small animal fracture healing model.
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- MATERIALS AND METHODS
Vascular volume calculated by 3D-HF-PDU showed a similar decline as that from microangiography from 2 to 8 weeks in Sham and OVX groups, similar to other studies of neovascularization in bone repair. For example, Raines et al.22 observed a decrease of neovascular volume in the marrow cavity from the peak value at 14 days after bone drilling in tibial marrow ablation rats. Zeng et al.23 found a high expression of vascular endothelial growth factor lasting from 14 to 28 days post-femoral fracture in rats; then it decreased gradually until 6 weeks.
We found an impaired angiogenic response in osteoporotic fracture healing. Other reports confirmed impaired revascularizaion in osteoporotic as compared to normal healing.1, 2 The mean signal intensity of blood flow is positively correlated to the concentration of moving blood cells.24 Risselada et al.'s data were similar to ours, in which the mean of signal scores of neovasculature, based on signal intensity peaked between 11 and 20 days post-fracture. A gradual decrease was seen thereafter.13 By utilizing the laser Doppler flowmetry, an optical in vivo imaging technique, Zheng et al.25 reported an increase in blood flow intensity from 2 to 4 weeks during bone defect healing in rabbits. The relative number of erythrocytes, calculated as the product of signal intensity and vascular volume, combines information of blood vessels and blood cells to evaluate local circulation.17, 26 We found a decrease of blood perfusion during callus mineralization and remodeling. The Sham group had better blood supply at the fracture site than the corresponding OVX group.
Due to the small sample size at each time, no significant correlation of vascular volume was found between 3D-HF-PDU and microangiography. However, all outcomes were pooled, a significant correlation was found. Our findings indicate 3D-HF-PDU is comparable to microangiography, and the results are consistent with previous studies. Also, 3D-HF-PDU is sensitive in distinguishing between normal and impaired angiogenic response. To our knowledge, this is the first study to assess changes in microvasculature and local blood flow during fracture healing in rats using 3D-HF-PDU. Currently, despite conventional microangiography being the gold standard for assessing microvasculature studies in fracture healing,6–8 it does not allow longitudinal analyses. Also, the results cannot provide flow information and can be affected by the quality of capillary perfusion due to fluctuations variables such as blood flush, perfusion pressure, and the threshold value chosen of the vascular tree during CT analysis.27 Conversely, 3D-HF-PDU is non-invasive, real-time, and can be used for longitudinal follow-up for both vascularization and blood flow quantifications.
In previous in vivo studies, investigators adopted power Doppler ultrasound to assess neovasculature at the fracture site in humans and large animals, detecting the existence of vascularity, evaluating the vessel area, vessel density, or blood flow intensity from 2D images.11–13, 28 However, since each 2D slice gives a different percentage of color pixels, many parallel slices must be measured throughout the total volume to reach a reliable result. Since 3D geometric evaluation can provide the spatial vascular tree, the value of vascular volume and blood flow within the VOI, 3D-HF-PDU measurement will improve the accuracy, objectivity, and integrity of the microcirculation information. Conventional frequency (2–15 MHz) ultrasound has difficulties in detecting small vessels (5–100 µm) and low flow velocities (∼0–50 mm/s). One feasible method is to raise the operating frequency to >20 MHz.29 Goertz et al.'s30 in vivo experiments confirmed that, at a center frequency of 50 MHz, the detection of vessels could be improved to 15–20 µm diameter in the mouse ear and demonstrated flow imaging in assessing a wide range of velocities (1–25 mm/s) in superficial mouse tumors. Therefore, we chose the transducer with the center frequency at 55 MHz, which was sensitive in detecting and assessing neovascularization around the fracture site.
There are some technical precautions for 3D-HF-PDU. First, the image noise must be reduced and filtered during scanning. Power Doppler is highly sensitive to motion artifacts that can be created by artery pulsations, breathing, or muscles contractions. An overly high Doppler gain, increasing the scan speed and vibration of the motor used in motor-steered probes may also generate disturbances. To reduce these artifacts, animals should be anesthetized with the extremities fixed, and a hand-free stand was utilized to mount the transducer for scanning. Also, it is appropriate to set the gain by up-regulating its value until random noise is encountered, then down-regulating until the noise disappears.31 The noise and low frequency flash artifacts could also be avoided by means of wall filters.32 Low filter settings can improve sensitivity, but may generate flash artifacts, whereas high filters can reduce the artifacts but will filter out flow in low levels. We found the wall filter at ∼2.5 mm/s to be suitable for scanning around the rat femur. Second, when dealing with the signals to assess microcirculation, appropriate imaging programs must be used to discriminate and filter large vessel signals. Finally, anesthesia will affect peripheral blood circulation, so each animal should be anesthetized with the same dose according to its body weight, placed at a set temperature, and positioned on a warming plate at that temperature to avoid variations caused by anesthesia. Complete scanning should be finished within a short time.
The application of 3D-HF-PDU has limitations. The detection of flow signals is limited by the penetration depth. With the use of higher Doppler frequency, penetration depth would be decreased, so researchers must compromise between accuracy and detection depth. We measured the muscle thickness at lateral and medial sides of the fracture site, which were 2.47 ± 0.33 and 4.06 ± 0.42 mm, respectively. The focal length of our transducer was 4.5 mm from the transducer surface in the soft tissues. Therefore, the resolution and penetration depth were adequate. Moreover, most sound waves were reflected as soon as they reached hard callus or cortical bone, so some vasculatures in the medullary cavity could not be detected, explaining why the absolute volume values detected by 3D-HF-PDU were consistently smaller than those of microangiography. There might be some overlap of the volume estimation depending on the size of the acoustic beam width. However, the beam width of a current ultrasound transducer is nearly identical to the scanning step size, so it will not significantly affect the comparison results.
Due to the advantages of small size (2–3 µm) and high backscatter that enhance the intensity of the signal, contrast agents have been developed to visualize vasculature at the capillary level, allowing a more sensitive measure of microcirculation.33, 34 We aimed to explore the feasibility of 3D-HF-PDU for assessing microvasculature, so we did not use contrast agents. However, for further improvement of image quality and more precise quantification, contrast agents are recommended for angiogenesis assessment at fractures in small animals.
In summary, our newly established technique using 3D-HF-PDU improved power Doppler 2D imaging to a 3D spatial view. Our results demonstrate the feasibility and reproducibility of 3D-HF-PDU for detecting and quantifying angiogenesis in fractured femurs of rats. Being non-invasive, while offering high-resolution anatomical visualization and objective data analysis, 3D-HF-PDU provides a robust approach for the evaluation of neovascular networks in the fracture healing of small animals, especially for in vivo longitudinal studies.