Angiogenesis plays an important role in various diseases such as cancer, atherosclerosis (vasa vasorum) and wound healing. Ultrasonic (US) imaging, super-resolution ultrasound, micro Doppler and acoustic angiography enable the imaging of vessel networks, but the information provided by these images is not quantitative. We propose real-time quantitative assessment of the microarchitecture of angiogenesis based on Ultrasound Multiple Scattering (USMS) analysis from raw ultrasound data. The proposed USMS parameters will enable the development of biomarkers of cancer aggressiveness, or of the probability of plaque rupture in atherosclerosis. We will validate our methods for cancer applications, but this novel biomedical imaging technology will find applications beyond cancer. For cancer diagnosis, US is highly sensitive but lacks specificity. However, X-Ray mammography can be ineffective in some populations and MRI present some challenges of cost, accessibility, and some contra-indications related to the use of Gadolinium contrast. A more specific US imaging technique would improve our ability to discriminate indeterminate non-lethal disease from lethal cancers, and make a clinical impact on a notable percentage of the at-risk population. Studies assessing the prognostic value of tumor angiogenesis have found a positive association between increasing microvessel densities and worsening prognosis. Several in-vivo studies with contrast enhanced US have demonstrated a relationship between microvascular structure and malignancy. There is a relationship between multiple scattering parameters, (such as the scattering mean free path and the anisotropy of the scattering mean free path), and micro-architectural parameters of the vasculature, (such as vessel density and anisotropy). We hypothesize that measuring parameters related to multiple scattering will increase the specificity of ultrasound for cancer diagnosis. To verify this hypothesis, we propose to develop, optimize and validate novel ultrasound methods utilizing multiple scattering approaches. These methods will enable the quantitative characterization of the micro-architecture of angiogenesis, leading to improvements in ultrasound specificity. In this project, we will: - Develop and optimize a quantitative ultrasound method to assess microvascular density and anisotropy non-invasively. - Establish the proof of concept that multiple scattering can be used to characterize angiogenesis and assess tumor malignancy in two rodent studies. - Validate our new technology in a preliminary study on human patients, and compare the diagnostic power of the developed methods to the currently available standard point of care ultrasound. This will enable us to verify the hypothesis that using ultrasound multiple scattering can increase the specificity of ultrasound for breast cancer diagnosis. If successful, this research will dramatically increase the specificity of US imaging of angiogenesis, and enable Contrast-Enhanced Ultrasound (CEUS) to become a reliable and widely used clinical tool for the diagnosis of cancer, or the evaluation of likelihood of plaque rupture. Ultimately, the methods developed will be used for screening, diagnosis and monitoring of lesions and atherosclerotic plaque.

We propose to develop novel ultrasound-based methods for the detection and quantification of pulmonary edema due to congestive heart failure (CHF). Ultrasound-based techniques are unsuitable for imaging the lung, due to the large amount of ultrasound wave multiple scattering from the millions of air-liquid interfaces due to alveolar walls and air filled alveoli. We propose to take advantage of this feature. Indeed, each scattering event can be seen as an opportunity for the ultrasound wave to embed information on the micro-architecture of the lung parenchyma. One of us has developed novel software algorithms to calculate the scattering mean free path (SMFP) of ultrasound waves propagated in lung tissue. We hypothesize that the scattering of ultrasound waves by the alveoli can be exploited to characterize the lung parenchyma. Measured SMFP will quantify edema in lung tissue. Three methods are currently used to image lungs to diagnose and follow lung disease: chest X-rays (CXR), CT scan, and nuclear magnetic resonance (NMR) scan. None of these imaging modalities can provide useful information about improving or worsening pulmonary congestion due to CHF. Serial ultrasound lung assessments (not imaging) would be particularly helpful for evaluation of progression or resolution of pulmonary edema due to CHF. We hypothesize that ultrasound scattering can be exploited to characterize the lung parenchyma, and quantify pulmonary edema. This novel hypothesis will be explored by accomplishing these specific aims: Aim 1. To develop ultrasonic methods for the assessment and staging of edema in a rat model of pulmonary edema by using a lung hilar clamp model creating ischemia-reperfusion injury that reliably results in pulmonary edema. Ultrasound methods exploiting the presence of multiple scattering will be implemented in in-vivo edematous and normal lungs. Wet:dry weight ratio (W/D), CT scans, and inflation-fixed lung histology will be used to quantify pulmonary edema. Changes in multiple scattering of ultrasound waves in lung due to edema is not related to the type of edema and protein content. Aim 2. To develop ultrasonic methods for the assessment of pulmonary edema in human lungs. The ultrasonic methods developed in Aim 1 will need to be adapted for use in the human lung by reducing frequency from 8 MHz to 3 to 5 MHz. Aim 3. To validate the proposed methods for quantification of pulmonary edema in human patients. Ten CHF patients will be studied after admission for a CHF exacerbation. Ultrasound parameters will be measured at 5 intercostal spaces on each side serially over 3-5 days and compared to other clinical parameters of pulmonary edema and to ultrasound parameters of normal lung obtained from 5 healthy volunteers. This translational project is responsive to this RFA because it pairs a bioengineer with two clinician-scientists to develop innovative, new, inexpensive methods to quantify pulmonary edema associated with CHF. Future large multicenter studies could be performed to assess utility of an inexpensive novel technology to monitor patients with CHF.

The objective of this research is to develop and validate novel ultrasound-based methods for the detection and localization of pulmonary nodules during minimally invasive thoracic surgery - Video Assisted Thoracic Surgery (VATS), and robotic surgery. Lung cancer is the leading cause of cancer deaths in the U.S., with over 220,000 new cases diagnosed each year. VATS and robotic surgery are performed to resect a pulmonary nodule, for diagnosis and therapeutic purposes. Pulmonary nodules are detected by CT scanning. However, during the surgery, pulmonary nodules can be extremely difficult to locate precisely. Small nodules might be extremely difficult to feel. Therefore, there is no guarantee that the nodule will be in the resected region of the lung parenchyma. This results in positive margins and sometimes a complete miss of the tumor in the resected lung wedge. The objective of this research is to develop novel ultrasound sequences, specific to the lung, exploiting the very complex properties of ultrasound multiple scattering in the lung parenchyma. Our approach is based on the following paradigm: Because pulmonary nodules are not filled with alveoli, they will not be responsible for as much multiple scattering as the healthy parenchymal tissue. We propose that mapping the amount of multiple scattering in the parenchyma will enable us to map pulmonary nodules in real time. We will develop ultrasound sequences and signal processing methods to quantify the amount of multiple scattering locally. In the future, the developed sequences will be implemented on an ultrasound probe placed in a flexible bronchoscope, which will be used during surgery to locate and ensure proper resection of pulmonary nodules.

This project will develop novel non-invasive ultrasound-based methods to quantify severity of pulmonary fibrosis. The objective is to prove that ultrasound can quantify severity of pulmonary fibrosis in a rodent model exhibiting varying degrees of pulmonary fibrosis, to justify future clinical studies in humans. This will be done by unducing pulmonary fibrosis in rats, and by developing novel ultrasound sequences measuring the diffusion of ultrasound waves in the parenchyma, which are expected to be related to the amount of fibrotic tissue and therefore to the severity of fibrosis. The developed methods will enable early diagnosis of pulmonary fibrosis, as well as serial monitoring of the response to treatments. This novel innovative technology can be viewed as a biomarker for pulmonary fibrosis, and can be used to evaluate therapeutic effects for patients with pulmonary fibrosis who are being treated.

Chronic wounds affect over 6.5 million patients in the United States alone and result in health costs of over $25 billion annually. Aging, obesity and diabetes increase the risks of non-healing wounds, and given the aging population and rise in diabetes and obesity, the healthcare burden associated with chronic wounds is only expected to increase in coming years. These statistics highlight that improper wound healing is a major and intensifying threat to both global health and the economy; unfortunately, because chronic wounds are frequently associated with a comorbid condition, the threat of is condition to public health and the economy is often overlooked. Proper wound healing is the result of a large number of interrelated biological events, which are orchestrated temporally in response to the injury microenvironment. Importantly, mechanical stimulation is a central cue to orchestrating cell behaviors during wound healing including proliferation, migration and extracellular matrix (ECM) production. Immediately following injury, a clot is formed which involves the formation of a platelet plug embedded within a fibrin mesh. Platelets bind multiple fibrin fibers and actively apply forces to contract the network, thereby stabilizing the developing clot. Furthermore, platelet-mediated clot contraction is thought to augment wound healing following cessation of bleeding by providing mechanical stimulation to surrounding cells. We have designed highly deformable platelet-like particles (PLPs) that mimic this clot contraction feature of native platelets by imparting high-affinity, high-specificity interactions with fibrin polymers (but not the soluble precursor fibrinogen). To maximize specific interactions with fibrin networks, we utilize highly deformable, ultra-low crosslinked Poly(N-isopropylacrylamide) (pNIPAm) hydrogel microparticles (���������gels) displaying multiple chemoligation sites as the base material for PLPs. To impart fibrin polymer specificity to the ���������gels, humanized synthetic single domain variable fragment (sdFv) antibodies with high affinity for fibrin, but not fibrinogen, were identified through molecular evolutionary techniques and then coupled to the ���������gels. These PLPs are capable of recapitulating a number of functions of native platelets, including augmentation of clotting of human plasma in vitro and decreasing bleeding times in rodent bleeding models. Our recently published study in Nature Materials shows that the high deformability of the ���������gels allows the PLPs to contract clots reminiscent of native platelets. In this application, we utilize ultrasound to enhance PLP-mediated clot contraction to delivery mechanical stimulation to cells in a chronic wound site to stimulate wound healing. This approach is paradigm shifting for treatment of chronic wounds; few strategies attempt to engage the mechanically sensitive pathways critical for proper wound healing, and those that do cannot deliver micromechanical stimulation in a controlled manner. Examples of important mechanically sensitive pathways in wound healing include Rho GTPase signaling, actin cytoskeleton engagement and mechanical activation of transforming growth factor beta (TGF��������), which leads to fibroblast activation and myofibroblast differentiation. The PLP technology described herein induces mechanical stimulation through a collective Brownian wrench mechanism to collapse the local matrix, inducing both global and cell-scale deformations. This tunable response allows for precise control over delivery of mechanical cues and provides the ability to supply stimulation at levels that promote healing without fibrosis. The overall objective of this proposal is to augment wound healing by combining PLP-mediated clot contraction with ultrasound therapy. Our central hypothesis is that ultrasound will accelerate PLP-mediated clot contraction in a well-controlled manner by increasing interactions between PLPs and fibrin networks, and significantly improve wound healing by providing mechanical stimulation to surrounding cells. In this proposal we will first

Atherosclerosis is the leading cause of morbidity and mortality in Western countries, in large part due to plaque rupture. Vulnerable plaques are the most likely to rupture, being the major cause of acute myocardial infarction and sudden cardiac death. Neovascularization of the arterial walls by adventitial vasa vasorum appears to participate in the progression of atherosclerosis and atherosclerotic plaque neovascularization was shown to be one of the strongest predictors of future cardiovascular events. No techniques are currently available for the quantitative assessment of vulnerable plaque, which remains an unmet need. Microbubbles are extremely powerful contrast agents for ultrasonic imaging of the vasculature and Contrast Enhanced IntraVascular UltraSound (CE-IVUS) seems to be one of the most promising imaging modalities for the detection of atherosclerosis and vulnerable plaque. However, although this technique allows the detection of the vasa vasorum, CE-IVUS currently does not provide a quantitative characterization of its micro-architecture. The specificity of CE-IVUS has to be improved. A quantitative assessment of the vasa vasorum network in vivo would strongly predict cardiovascular events yet no such techniques currently exist. We propose using ultrasound multiple scattering to characterize the microstructural properties of neovascular networks. By using ultrasound multiple scattering to characterize the architecture of the neovascularization, we will add a new dimension to CE-IVUS. With the technique we propose to develop here, CE-IVUS will not only be used for the detection of plaque related neo-angiogenesis, but also for its characterization. This will dramatically increase the specificity of CE-IVUS for the assessment of plaque vulnerability. It will enable the ultrasonic assessment of the vasa vasorum to become a widely used clinical tool for screening, diagnosis and monitoring of plaque vulnerability.

Osteoporosis is increasingly prevalent. It modifies microarchitecture and bone density and the assessment of both is required to predict bone competence accurately. Several studies have demonstrated the relationship between bone micro-architecture and bone strength. However, the current gold standard for fracture risk assessment relies on the X-ray characterization of bone mineral density alone. A quantitative, non-invasive and non-ionizing characterization of bone micro-architecture is currently not possible in vivo. There is therefore an unmet need. We propose to address this need by developing a novel technique for the assessment of bone micro-architecture using multiple scattering of ultrasound. We hypothesize that when multiple scattering occurs there is a measureable relationship between ultrasonic parameters, such as the scattering mean free path, and micro-architectural parameters, such as anisotropy, connectivity and trabecular separation. Our approach is based on the following two-fold paradigm: First: Combining the characterization of bone micro-architecture to the currently available assessment of bone mineral density would improve the diagnosis of fracture risk. Second: Ultrasound waves in the MHz range are subjected to multiple scattering by the micro-structure during propagation in bone. The resulting ultrasonic signals are complex and embed information on the micro-architecture. We will have the following specific aims: SA.1 We will establish a quantitative relationship between the micro-architectural properties of bone and ultrasound parameters related to multiple scattering. This will be validated in vitro in bone phantoms and real specimen. SA.2 Using mechanical testing, we will investigate the relationship between ultrasound parameters, micro-architecture and mechanical competence. SA.3 We will develop a measurement strategy for the in vivo characterization of bone micro-architecture, based on the relationships developed in SA 1 and SA2. If successful, this research will lead to a quantitative, non-invasive and non-ionizing ultrasound-based method to characterize bone micro-architecture. Ultimately, the methods developed will be used for screening, diagnosis and monitoring of osteoporosis. This research aims at limiting the use of ionizing and costly techniques for the diagnosis of osteoporosis.

Osteoporosis modifies the microarchitecture of trabecular bone by increasing the trabecular spacing and the anisotropy. Assessing these parameters will improve the assessment of bone mechanical competence, and will allow the prevention of spontaneous vertebral fractures. A quantitative, non-invasive and non-ionizing characterization of bone micro-architecture is currently not possible in vivo. The ultrasonic characterization of the biomechanical properties of bone has the potential to improve fracture risk assessment with a non-invasive, relatively inexpensive method that doesn't rely on ionizing radiation. Several independent research results suggest that multiple scattering of ultrasonic waves by bone heterogeneities plays an important role in the bone-ultrasound interaction. We propose to develop ultrasonic techniques that characterize bone micro-architecture based on multiple scattering. Our objectives for this project is to develop ultrasonic techniques for the measurement of bone micro-architecture on bone samples in vitro. The results will be validated by comparing the parameters measured with ultrasound (trabecular spacing and anisotropy) to the actual parameters of the micro-architecture measured with X-Ray Computed Tomography.

Our objective is to improve the diagnosis and monitoring of spaceflight osteopenia through the determination of micro-structure of trabecular bone from ultrasonic measurements. In spaceflight osteopenia, the absence of gravity leads to reduced stresses on the body, which is believed to impair the process of bone remodelling. As a consequence, bone loss is experienced by astronauts, as well as modifications of the trabecular micro-architecture. Ultrasound waves in the MHz range are subjected to multiple scattering by the trabeculae during propagation in bone. The resulting ultrasonic signals are complex and embed information on the micro-architecture. We propose to test the hypothesis that when multiple scattering occurs there is a measureable relationship between ultrasonic parameters and micro-architectural parameters, such as trabecular spacing and anisotropy. To determine this relationship we will establish a model of ultrasonic propagation in trabecular bone based on the scattering theory of elastic waves in a complex heterogeneous environment. We will identify a set of ultrasound parameters quantitatively related to specific micro-architectural properties such as anisotropy, trabecular spacing and connectivity. We will develop a practical, model-based strategy for the monitoring of bone micro-architectural properties during space flight.