Fabricating Blood Vessels with Ultrasound
Diane Dalecki, Ph.D.
Eric S. Comeau, M.S.
Denise C. Hocking, Ph.D.
Rochester Center for Biomedical Ultrasound
University of Rochester
Rochester, NY 14627
Popular version of paper 3aBA5, “Applications of acoustic radiation force for microvascular tissue engineering”
Presented Wednesday morning May 20, 9:25 AM, in room Kings 2
169th ASA Meeting, Pittsburgh
Tissue engineering is the field of science dedicated to fabricating artificial tissues and organs that can be made available for patients in need of organ transplantation or tissue reconstructive surgery. Tissue engineers have successfully fabricated relatively thin tissues, such as skin substitutes, that can receive nutrients and oxygen by simple diffusion. However, recreating larger and/or more complex tissues and organs will require developing methods to fabricate functional microvascular networks to bring nutrients to all areas of the tissue for survival.
In the laboratories of Diane Dalecki, Ph.D. and Denise C. Hocking, Ph.D., research is underway to develop new ultrasound technologies to control and enhance the fabrication of artificial tissues1. Ultrasound fields are sound fields at frequencies higher than humans can hear (i.e., > 20 kHz). Dalecki and Hocking have developed a technology that uses a particular type of ultrasound field, called an ultrasound standing wave field, as a tool to non-invasively engineer complex spatial patterns of cells2 and fabricate microvessel networks3,4 within artificial tissue constructs.
When a solution of collagen and cells is exposed to an ultrasound standing wave field, the forces associated with the field lead to the alignment of the cells into planar bands (Figure 1). The distance between the bands of cells is controlled by the ultrasound frequency, and the density of cells within each band is controlled by the intensity of the sound field. The collagen polymerizes into a solid gel during the ultrasound exposure, thereby maintaining the spatial organization of the cells after the ultrasound is turned off. More complex patterning can be achieved by use of more than one ultrasound transducer.
Figure 1. Acoustic-patterning of microparticles (dark bands) using an ultrasound standing wave field. Distance between planar bands is 750 µm. Scale bar = 100 μm
An exciting application of this technology involves the fabrication of microvascular networks within artificial tissue constructs. Specifically, acoustic-patterning of endothelial cells into planar bands within collagen hydrogels leads to the rapid development of microvessel networks throughout the entire volume of the hydrogel. Interestingly, the structure of the resultant microvessel network can be controlled by choice of the ultrasound exposure parameters. As shown in Figure 2, ultrasound standing wave fields can be employed to fabricate microvessel networks with different physiologically relevant morphologies, including capillary-like networks (left panel), aligned non-branching vessels (center panel) or aligned vessels with hierarchically branching microvessels. Ultrasound fields provide an ideal technology for microvascular engineering; the technology is rapid, noninvasive, can be broadly applied to many types of cells and hydrogels, and can be adapted to commercial fabrication processes.
To learn more about this research, please view this informative video (https://www.youtube.com/watch?v=ZL-cx21SGn4).
Figure 2. Ultrasound-fabricated microvessel networks within collagen hydrogels. The ultrasound pressure amplitude used for initial patterning determines the final microvessel morphology, which can resemble torturous capillary-like networks (left panel), aligned non-branching vessels (center panel) or aligned vessels with hierarchically branching microvessels. Scale bars = 100 μm.
 Dalecki D, Hocking DC. Ultrasound technologies for biomaterials fabrication and imaging. Annals of Biomedical Engineering 43:747-761; 2015.
 Garvin KA, Hocking DC, Dalecki D. Controlling the spatial organization of cells and extracellular matrix proteins in engineered tissues using ultrasound standing wave fields. Ultrasound Med. Biol. 36:1919-1932; 2010.
 Garvin KA, Dalecki D, Hocking DC. Vascularization of three-dimensional collagen hydrogels using ultrasound standing wave fields. Ultrasound Med. Biol. 37:1853-1864; 2011.
 Garvin KA, Dalecki D, Youssefhussien M, Helguera M, Hocking DC. Spatial patterning of endothelial cells and vascular network formation using ultrasound standing wave fields. J. Acoust. Soc. Am. 134:1483-1490; 2013.
The clinical evaluation of solid tumors typically includes needle biopsies, which can provide diagnostic (benign vs. cancer) and molecular information (targetable mutations, drug resistance, etc). This procedure has several diagnostic limitations, most notably, the potential to miss the mutations only millimeters away. In response to these limitations, the concept of “liquid biopsy” has emerged in recent years: the detection of nucleic acid cancer biomarkers, such as tumor-derived microRNAs (miRNAs) and circulating tumor DNA (ctDNA). These biomarkers have shown high diagnostic value and could guide the selection of appropriate targeted therapies. However, the abundance of these biomarker classes in the circulation is often too low to be detectable even with the most sensitive techniques because of their low levels of release from the tumor.
How can we make tumor cells release these biomarkers into the blood? The most straightforward way would be to puncture the cell membrane so that its content is released. One technology that allows for just that is high intensity focused ultrasound (HIFU). HIFU uses powerful, controlled ultrasound waves that are focused inside human body to ablate the targeted tissue at the focus without affecting the surrounding organs. Alternatively, if HIFU waves are sent in short, infrequent but powerful bursts, they cause mechanical disruption of tissue at the focus without any thermal effects. The disruption is achieved by small gas bubbles in tissue that appear, grow and collapse in response to the ultrasound wave– a phenomenon known as cavitation. Depending on the pulsing protocol employed, the outcome can range from small holes in cell membranes and capillaries to complete liquefaction of a small region of tumor. Using this technology, we seek to release biomarkers from tumor cells into the circulation in the effort to detect them using a blood test and avoiding biopsy.
Figure caption: Experimental setup and the basic concept of “ultrasound-aided liquid biopsy”. Pulsed high intensity focused ultrasound (HIFU) waves create, grow and collapse bubbles in tissue, which leads to puncturing of cell membranes and capillary walls. Cancer-derived microRNAs are thus released from the cells into the circulation and can be detected in a blood sample.
To test this approach, we applied pulsed HIFU exposures to prostate cancer tumors implanted under the skin of laboratory rats, as illustrated in the image above. For image guidance and targeting we used conventional ultrasound imaging. Blood samples were collected immediately before and at periodic intervals after HIFU treatment and were tested for the presence of microRNAs that are associated with rat prostate cancer. The levels of these miRNAs were elevated up to 12-fold within minutes after the ultrasound procedure, and then declined over the course of several hours. The effects on tissue were evaluated in the resected tumors, and we found only micron-sized areas of hemorrhage scattered through otherwise intact tissue, suggesting damage to small capillaries. These data provided the proof of principle for the approach that we termed “ultrasound-aided liquid biopsy”. We are now working on identifying other classes of clinically valuable biomarkers, most notably tumor-derived DNA, that could be amplified using this methodology.
Tatiana Khokhlova – email@example.com
George Schade – firstname.lastname@example.org
Yak-Nam Wang – email@example.com
Joo Ha Hwang – firstname.lastname@example.org
University of Washington
1013 NE 40th St
Seattle, WA 98105
John Chevillet – email@example.com
Institute for Systems Biology
401 Terry Ave N
Seattle, WA 98109
Maria Giraldez – firstname.lastname@example.org
Muneesh Tewari – email@example.com
University of Michigan
109 Zina Pitcher Place 4029
Ann Arbor, MI 48109
Popular version of paper 4pBA1
Presented Thursday afternoon, October 30, 2014, at 1:30 pm
168th ASA Meeting, Indianapolis
The cervix, which is the opening into the uterus, is a remarkable organ. It is the only structure in the entire body that has diametrically opposed functions. The normal cervix stays stiff and closed throughout most of pregnancy while the baby develops and then completely softens and opens at just the right time so the full-grown baby can be born normally, through the vagina. This process of softening and opening involves remodeling, or breaking down, of the cervix’s collagen structure together with an increase in water, or hydration, of the cervix. When the process happens too soon, babies can be born prematurely, which can cause serious lifelong disability and even death. When it happens too late, mothers may need a cesarean delivery. [1,2]
Amazingly, even after 100 years of research, nobody understands how this happens. Specifically, it is unclear how the cervix knows when to soften, or what molecular signals initiate and control this process. As a result, obstetrical providers today evaluate cervical softness in exactly the same way as they did in the 1800s: they feel the cervix with their fingers and classify it as ‘soft’, ‘medium’ or ‘firm’. And, unsurprisingly, despite significant research effort, the preterm birth rate remains unacceptably high and more than 95% of preterm births are intractable to any available therapies.
The cervix is like a cylinder with a canal in the middle that goes up into the uterus. This is where the sperm travels to fertilize the egg, which then nests in the uterus. Cervical collagen is roughly arranged in 3 layers. There is a thin layer on the inner part that where collagen is mostly aligned along the cervical canal, and similarly a thin layer that runs along the outside of the cervix. Between these layers, in the middle of the cervix is a thicker layer of collagen that is circumferential, aligned like a belt around the canal. Researchers theorize that the inner and outer layers stabilize the cervix from tearing off as it shortens and dilates for childbirth, while the middle circumferential layer is most important for the strength and stiffness of the cervix to prevent it from opening prematurely, but nobody is certain. This information is critical to understanding the process of normal, and abnormal, cervical remodeling, and that is why recently there is growing interest in developing technology that can non-invasively and thoroughly assess the collagen in the cervix and measure its softness and hydration status. The most promising approaches use regular clinical ultrasound systems that have special processing strategies to evaluate these tissue properties.
One approach to assessing tissue softness uses ultrasound pressure to induce a shear wave, which moves outward like rings in water after a stone is dropped. The speed of the shear wave provides a measurement of tissue stiffness because these waves travel slower in softer tissue. This method is used clinically in simple tissues without layers of collagen, such as the liver for staging fibrosis. Cervical tissue is very complex because of the collagen layers, but, after many years of research, we finally have adapted this method for the cervix. The first thing we did was compare shear wave speeds in hysterectomy specimens (surgically removed uterus and cervix) between two groups of women. The first group was given a medicine that softens the cervix in preparation for induction of labor, a process called “ripening”. The second group was not treated. We found that the shear wave speeds were slower in the women who had the medicine, indicating that our method could detect the cervices that had been softened with the medicine. We also found that the cervix was even more complex than we’d thought, that shear wave speeds are different in different parts of the cervix. Fortunately, we learned we could easily control for that by measuring the same place on every woman’s cervix. We confirmed that our measurements were accurate by performing second harmonic generation microscopy on the cervix samples in which we measured shear wave speeds. This is a sophisticated way of looking at the tiny collagen structure of a tissue. It told us that indeed, when the collagen structure was more organized, the shear wave speeds were faster, indicating stiffer tissue, and when the collagen started breaking down, the shear wave speeds were slower, indicating softer tissue.
The next step was to see if our methods worked outside of the laboratory. So we studied pregnant women who were undergoing cervical ripening in preparation for induction of labor. We took measurements from each woman before they got the ripening medicine, and then afterwards. We found that the shear wave speeds were slower after the cervix had been ripened, indicating the tissue had softened. This told us that we could obtain useful information about the cervix in pregnant women.
We also evaluated attenuation, which is loss of ultrasound signal as the wave propagates, because attenuation should increase as tissue hydration increases. This is a very complicated measurement, but we found that if we carefully control the angle of the ultrasound beams relative to the underlying cervical structure, attenuation estimates are sensitive to the difference between ripened and unripened tissue in hysterectomy specimens. This suggests the potential for an additional parameter to quantify and monitor the status of the cervix during pregnancy, and we are currently analyzing attenuation in the pregnant women before and after they received the ripening medicine.
This technology is exciting because it could change clinical practice. On a very basic level, obstetrical providers would be able to talk in objective terms about a woman’s cervix instead of the subject “soft, medium, or firm” designation they currently use, which would improve provider communication and thus patient care. More importantly, it could provide a means to thoroughly study normal and abnormal cervical remodeling, and associate structural changes in the cervix with molecular changes, which is the only way to discover new interventions for preterm birth.
1. Feltovich H, Hall TJ, and Berghella V. Beyond cervical length: Emerging technologies for assessing the pregnant cervix. Am J Obst & Gyn, 207(5): 345-354, 2012.
2. Feltovich, H and Hall TJ. Quantitative Imaging of the Cervix: Setting the Bar. Ultrasound Obstet Gynecol. 42(2): 121-128, 2013.
3. Carlson LC, Feltovich H, Palmeri ML, Dahl JJ, Del Rio AM, Hall TJ, Estimation of shear wave speed in the human uterine cervix. Ultrasound Obstet Gynecol. 43( 4): 452–458, 2014.
4. Carlson LC, Romero ST, Palmeri ML, Munoz del Rio A, Esplin SM, Rotemberg VM, Hall TJ, Feltovich H. Changes in shear wave speed pre and post induction of labor: A feasibility study. Ultrasound Obstet Gynecol.published online ahead of print Sep.2014.
Unlocking the Mystery of the Cervix
Timothy J Hall, Helen Feltovich, Lindsey C. Carlson, Quinton W. Guerrero, Ivan M. Rosado-Mendez
University of Wisconsin, Madison, WI (firstname.lastname@example.org)
During the civil war, the risk of lethal infection drove surgeons to perform multiple amputations on wounded soldiers. The loss of life from the infection outweighed the loss of the limb. In modern medicine, the occurrence of amputations is much less due to the development of sterile surgical techniques, but a type of “amputation” is still the only treatment option for many patients battling infection.
In modern medicine, numerous implants have been developed to treat many different ailments ranging from basic hernia, to pacemakers, to neuronal implants to control seizures. These implants play a vital role in the restoration of function or quality of life for these patients. However, if an infection grows on the implant despite sterile surgical techniques, then the only treatment option is to remove and replace the infected implant with a new device. The bacteria responsible for the infection protect themselves by forming a biofilm on the surface of the implant. Bacteria in the biofilm are protected from antibiotics and the administration of antibiotics can even cause the formation of antibiotic resistant strains. Recently, however, we have shown that focused ultrasound can precisely target and destroy these biofilms (Figure 1). Therefore, in the future, we hope to develop a noninvasive therapy to treat infections on medical implants based on ultrasound.
Fig 1: The surface of graphite plates after growing Pseudomonas aeruginosa biofilms and exposing to high-intensity focused ultrasound. Green shows live cells while red shows dead cells. In the absence of treatment, a live biofilm is clearly visible. The ultrasound exposures resulted in almost complete biofilm destruction with few if any live cells remaining.
There are two primary types of therapy that can be performed with ultrasound. The first uses the energy in the sound to heat the tissue. The second uses the sound to excite microscopic bubbles in the tissue resulting in a mechanical change to the tissue structure. Our technology is based on the generation and subsequent excitation of the microscopic bubbles. The high-intensity of the sound causes the bubbles to violently collapse shredding cells adjacent to the bubbles. In addition to treating biofilm infections, we have also shown that the excitation of these microscopic bubbles can lyse microalgae for the release of lipids. These lipids can then be utilized in the formation of biofuels. The use of focused ultrasound was shown to be more energy efficient than other comparable methods of lysing the microalgae.
Timothy A Bigelow – email@example.com
Iowa State University
2113 Coover Hall
Ames, IA 50014
Popular version of paper 4pBA3
Presented Thursday afternoon, October 30, 2014
168th ASA Meeting, Indianapolis
Stone disease in the children is becoming more commonplace. Over the past 25 years the incidence has increased approximately 6-10% annually and is now 50 per 100,000 adolescents 1. The diagnosis of kidney stones in children, as in adults, relies primarily on diagnostic imaging. In adults, the most common imaging study performed in the United States for the initial diagnosis of kidney stones is a computed tomography (CT) scan, due to its superior sensitivity and specificity. This is not preferred for children as CT utilizes ionizing radiation and children have an increased sensitivity to radiation effects. In addition, stone formers often have recurrent stone episodes over their lifetime, which is especially relevant to younger stone formers. The repeated exposures of a CT scan could lead to an increased risks of secondary cancers2. As a result, ultrasound is often performed in children instead because there is no radiation associated with its use1. Ultrasound, however, is less sensitive and specific compared with CT, and is known to overestimate kidney stone size3-5, which is one of the primary determinants of how stones are managed.
We have identified a new technique to improve the accuracy of US stone sizing. Traditionally, a kidney stone will show up as a bright, or hyperechoic, object on US, and radiologists measure the longest linear dimension of the bright area to represent the stone size. We believe that the dark, or hypoechoic, acoustic shadow that appears behind a kidney stone provides additional information for predicting stone size. The resolution of the stone is affected by the distortion of the waves traveling through the intervening tissues; the resolution of the shadow is only affected by the local stone obstruction.
We screened 660 stone diagnoses over an 11 year period (2004 – 2014) at Seattle Children’s Hospital, a tertiary care referral center serving the greater Pacific Northwest. Over the study period, there were 37 patients presenting with an initial diagnosis of a kidney stone, and who had both a US and CT within three months of each other. Two reviewers retrospectively measured both the stone size and the shadow width from the ultrasound image. We compared the results to the stone size measured from the CT scan. A total of 48 stones were included in the study with an average size based on CT imaging of 7.85 mm. The shadow width was present in 88% of the cases, and, on average, was more accurate than measuring the stone itself. Measuring the stone width on ultrasound tended to overestimate the size of the stone by 1.2 ± 2.5 mm (reviewer 1) and 2.0 ± 1.7 mm (reviewer 2), while measuring the shadow width on ultrasound underestimated the size of the stone by -0.6 ± 2.5 mm (reviewer 1) and overestimated the stone size by 0.3 ± 1.2 mm (reviewer 2). In both cases, the shadow was a better predictor of stone size and, for reviewer 2, there was less variability in the data. Stone sizes based on CT are typically considered within 1 mm, with low variability.
Our technique is simple and can be easily adopted by pediatricians, radiologists, and urologists. It improves the accuracy of US and gives physicians more confidence that the reported size is more representative of the true stone size, without having to expose children to the radiation of a CT scan. This also allows the physician to make more accurate decisions about when to perform a surgery for a large stone or continue to observe a small stone that may pass on its own. The findings from this study may also potentially be applicable to stones in adult patients. We believe that with continued advancements in US, we can reduce the number of CT scans for both adults and children. The results in part highlight one of the drawbacks of ultrasound, which is user dependence; two users can have very different results. It is anticipated that future work on automated stone and shadow sizing can reduce this along with standardizing the method in which the shadow measurement is taken.
1. Tasian G and Copelovitch L. Evaluation and Management of Kidney Stones in Children. J Urol. 2014: Epub ahead of print
2. Pearce MS, Salotti JA, Little MP, et al. Radiation exposure from CT scans in childhood and subsequent risk of leukaemia and brain tumours: a retrospective cohort study. Lancet 2012; 380: 499-505.
3. Ray AA, Ghiculete D, Pace KT, Honey RJD. Limitations to ultrasound in the detection and measurement of urinary tract calculi. Urology 2010; 76(2):295-300.
4. Fowler KAB, Locken JA, Duchesne JH, Williamson MR. US for detecting renal calculi with nonehnanced CT as a reference standard. Radiology 2002; 222(1):109-113.
5. Dunmire B, Lee FC, Hsi RS, Cunitz BW, Paun M, Bailey MR, Sorensen MD, Harper JD. Tools to improve the accuracy of kidney stone sizing with ultrasound. Aug 2014; [Epub ahead of print].
Franklin C. Lee1 – firstname.lastname@example.org
Jonathan D. Harper 1 – email@example.com
Thomas S. Lendvay1, 2 – Thomas.firstname.lastname@example.org
Ziyue Liu3 – email@example.com
Barbrina Dunmire 4 – firstname.lastname@example.org
Manjiri Dighe 5 – email@example.com
Michael Bailey – firstname.lastname@example.org
Mathew D. Sorensen1, 6 – email@example.com
University of Washington
1 Department of Urology, Box 356510
5 Department of Radiology, Box 357115
1959 NE Pacific St, Seattle, WA 98195
2 Seattle Children’s Hospital
Urology, Developmental Pediatrics
4800 Sand Point WA NE, Seattle, WA 98105
3 Indiana University
Department of Biostatistics
410 W. Tenth St, Suite 3000, Indianapolis, IN 46202
4 University of Washington
Applied Physics Lab – Center for Industrial and Medical Ultrasound2
1013 NE 40th St, Seattle, WA 98105
6 Department of Veteran Affairs Medical Center
Division of Urology
1660 South Columbian Way, Seattle, WA 98108