Grand Challenges at the Interface of Engineering and Medicine.

Over the past two decades Biomedical Engineering has emerged as a major discipline that bridges societal needs of human health care with the development of novel technologies. Every medical institution is now equipped at varying degrees of sophistication with the ability to monitor human health in both non-invasive and invasive modes. The multiple scales at which human physiology can be interrogated provide a profound perspective on health and disease. We are at the nexus of creating "avatars" (herein defined as an extension of "digital twins") of human patho/physiology to serve as paradigms for interrogation and potential intervention. Motivated by the emergence of these new capabilities, the IEEE Engineering in Medicine and Biology Society, the Departments of Biomedical Engineering at Johns Hopkins University and Bioengineering at University of California at San Diego sponsored an interdisciplinary workshop to define the grand challenges that face biomedical engineering and the mechanisms to address these challenges. The Workshop identified five grand challenges with cross-cutting themes and provided a roadmap for new technologies, identified new training needs, and defined the types of interdisciplinary teams needed for addressing these challenges. The themes presented in this paper include: 1) accumedicine through creation of avatars of cells, tissues, organs and whole human; 2) development of smart and responsive devices for human function augmentation; 3) exocortical technologies to understand brain function and treat neuropathologies; 4) the development of approaches to harness the human immune system for health and wellness; and 5) new strategies to engineer genomes and cells.

FRET-Based Ca2+ Biosensor Single Cell Imaging Interrogated by High-Frequency Ultrasound.

Fluorescence resonance energy transfer (FRET)-based biosensors have advanced live cell imaging by dynamically visualizing molecular events with high temporal resolution. FRET-based biosensors with spectrally distinct fluorophore pairs provide clear contrast between cells during dual FRET live cell imaging. Here, we have developed a new FRET-based Ca2+ biosensor using EGFP and FusionRed fluorophores (FRET-GFPRed). Using different filter settings, the developed biosensor can be differentiated from a typical FRET-based Ca2+ biosensor with ECFP and YPet (YC3.6 FRET Ca2+ biosensor, FRET-CFPYPet). A high-frequency ultrasound (HFU) with a carrier frequency of 150 MHz can target a subcellular region due to its tight focus smaller than 10 µm. Therefore, HFU offers a new single cell stimulations approach for FRET live cell imaging with precise spatial resolution and repeated stimulation for longitudinal studies. Furthermore, the single cell level intracellular delivery of a desired FRET-based biosensor into target cells using HFU enables us to perform dual FRET imaging of a cell pair. We show that a cell pair is defined by sequential intracellular delivery of the developed FRET-GFPRed and FRET-CFPYPet into two target cells using HFU. We demonstrate that a FRET-GFPRed exhibits consistent 10-15% FRET response under typical ionomycin stimulation as well as under a new stimulation strategy with HFU.

Three-Dimensional Printed Piezoelectric Array for Improving Acoustic Field and Spatial Resolution in Medical Ultrasonic Imaging.

Piezoelectric arrays are widely used in non-destructive detecting, medical imaging and therapy. However, limited by traditional manufacturing methods, the array's element is usually designed in simple geometry such as a cube or rectangle, restricting potential applications of the array. This work demonstrates an annular piezoelectric array consisting of different concentric elements printed by Mask-Image-Projection-based Stereolithography (MIP-SL) technology. The printed array displays stable piezoelectric and dielectric properties. Compared to a traditional single element transducer, the ultrasonic transducer with printed array successfully modifies the acoustic beam and significantly improves spatial resolution.

Effects of CD49d-targeted antisense-oligonucleotide on α4 integrin expression and function of acute lymphoblastic leukemia cells: Results of in vitro and in vivo studies.

We recently demonstrated the effectiveness of blocking CD49d with anti-functional antibodies or small molecule inhibitors as a rational targeted approach to the treatment of acute leukemia in combination with chemotherapy. Antisense oligonucleotide promises to be no less specific than antibodies and inhibitors, but more interesting for pharmacokinetics and pharmacodynamics. We addressed this using the published CD49d antisense drug ATL1102. In vitro, we incubated/nucleofected the ALL cell line Kasumi-2 with ATL1102. In vivo, immunodeficient hosts were engrafted with primary ALL cells and treated with ATL1102. Changes in expression of CD49d mRNA and CD49d protein, and of cooperating gene products, including ß1 integrin and CXCR4, as well as survival in the mouse experiments were quantified. We observed dose-dependent down-regulation of CD49d mRNA and protein levels and its partner integrin ß1 cell surface protein level and, up-regulation of CXCR4 surface expression. The suppression was more pronounced after nucleofection than after incubation, where down-regulation was significant only at the higher doses. In vivo effects of ATL1102 were not sufficient to translate into "clinical" benefit in the leukemia model. In summary, antisense oligonucleotides are successful tools for specifically modulating gene expression but sufficient delivery to down-regulate CD49d in vivo may be difficult to achieve.

High-Frequency Ultrasonic Imaging with Lead-free (Na,K)(Nb,Ta)O3 Single Crystal.

Lead-free (Na,K)(Nb,Ta)O3 (KNNT) piezoelectric single crystal has been successfully grown using the top-seeded solution growth technique. The electromechanical coupling factors are very high ( k33 = 0.827, kt = 0.646), and the dielectric loss tangent is as low as 0.004. Acoustic impedance was calculated to be 26.5 MRayl. From the single crystal, a single element transducer was fabricated. The transducer achieved a 57.6% -6 dB bandwidth and 32.3 µm axial resolution at the center frequency of 45.4 MHz, which can identify the cornea of porcine eyeball with high resolution. Comparison between KNNT single crystal and lead-based single crystal was discussed. The results suggest that this single crystal transducer is an excellent candidate to replace lead-containing transducer for high-frequency ultrasonic imaging applications.

Ultrasonic Transducer-Guided Electrochemical Impedance Spectroscopy to Assess Lipid-Laden Plaques.

Plaque rupture causes acute coronary syndromes and stroke. Intraplaque oxidized low density lipoprotein (oxLDL) is metabolically unstable and prone to induce rupture. We designed an intravascular ultrasound (IVUS)-guided electrochemical impedance spectroscopy (EIS) sensor to enhance the detection reproducibility of oxLDL-laden plaques. The flexible 2-point micro-electrode array for EIS was affixed to an inflatable balloon anchored onto a co-axial double layer catheter (outer diameter = 2 mm). The mechanically scanning-driven IVUS transducer (45 MHz) was deployed through the inner catheter (diameter = 1.3 mm) to the acoustic impedance matched-imaging window. Water filled the inner catheter to match acoustic impedance and air was pumped between the inner and outer catheters to inflate the balloon. The integrated EIS and IVUS sensor was deployed into the ex vivo aortas dissected from the fat-fed New Zealand White (NZW) rabbits (n=3 for fat-fed, n= 5 normal diet). IVUS imaging was able to guide the 2-point electrode to align with the plaque for EIS measurement upon balloon inflation. IVUS-guided EIS signal demonstrated reduced variability and increased reproducibility (p < 0.0001 for magnitude, p < 0.05 for phase at < 15 kHz) as compared to EIS sensor alone (p < 0.07 for impedance, p < 0.4 for phase at < 15 kHz). Thus, we enhanced topographic and EIS detection of oxLDL-laden plaques via a catheter-based integrated sensor design to enhance clinical assessment for unstable plaque.

Role of Excessive Autophagy Induced by Mechanical Overload in Vein Graft Neointima Formation: Prediction and Prevention.

Little is known regarding the interplays between the mechanical and molecular bases for vein graft restenosis. We elucidated the stenosis initiation using a high-frequency ultrasonic (HFU) echogenicity platform and estimated the endothelium yield stress from von-Mises stress computation to predict the damage locations in living rats over time. The venous-arterial transition induced the molecular cascades for autophagy and apoptosis in venous endothelial cells (ECs) to cause neointimal hyperplasia, which correlated with the high echogenicity in HFU images and the large mechanical stress that exceeded the yield strength. The ex vivo perfusion of arterial laminar shear stress to isolated veins further confirmed the correlation. EC damage can be rescued by inhibiting autophagy formation using 3-methyladenine (3-MA). Pretreatment of veins with 3-MA prior to grafting reduced the pathological increases of echogenicity and neointima formation in rats. Therefore, this platform provides non-invasive temporal spatial measurement and prediction of restenosis after venous-arterial transition as well as monitoring the progression of the treatments.

Optimal flushing agents for integrated optical and acoustic imaging systems.

An increasing number of integrated optical and acoustic intravascular imaging systems have been developed and hold great promise for accurately diagnosing vulnerable plaques and guiding atherosclerosis treatment. However, in any intravascular environment, the vascular lumen is filled with blood, a high-scattering source for optical and high-frequency ultrasound signals. Blood must be flushed away to provide clearer images. To our knowledge, no research has been performed to find the ideal flushing agent for combined optical and acoustic imaging techniques. We selected three solutions as potential flushing agents for their image-enhancing effects: mannitol, dextran, and iohexol. Testing of these flushing agents was performed in a closed-loop circulation model and in vivo on rabbits. We found that a high concentration of dextran was the most useful for simultaneous intravascular ultrasound and optical coherence tomography imaging.

Ultrahigh frequency lensless ultrasonic transducers for acoustic tweezers application.

Similar to optical tweezers, a tightly focused ultrasound microbeam is needed to manipulate microparticles in acoustic tweezers. The development of highly sensitive ultrahigh frequency ultrasonic transducers is crucial for trapping particles or cells with a size of a few microns. As an extra lens would cause excessive attenuation at ultrahigh frequencies, two types of 200-MHz lensless transducer design were developed as an ultrasound microbeam device for acoustic tweezers application. Lithium niobate single crystal press-focused (PF) transducer and zinc oxide self-focused transducer were designed, fabricated and characterized. Tightly focused acoustic beams produced by these transducers were shown to be capable of manipulating single microspheres as small as 5 µm two-dimensionally within a range of hundreds of micrometers in distilled water. The size of the trapped microspheres is the smallest ever reported in the literature of acoustic PF devices. These results suggest that these lensless ultrahigh frequency ultrasonic transducers are capable of manipulating particles at the cellular level and that acoustic tweezers may be a useful tool to manipulate a single cell or molecule for a wide range of biomedical applications.

Integrated optical coherence tomography, ultrasound and photoacoustic imaging for ovarian tissue characterization.

Ovarian cancer has the lowest survival rate of the gynecologic cancers because it is predominantly diagnosed in Stages III or IV due to the lack of reliable symptoms, as well as the lack of efficacious screening techniques. Detection before the malignancy spreads or at the early stage would greatly improve the survival and benefit patient health. In this report, we present an integrated optical coherence tomography (OCT), ultrasound (US) and photoacoustic imaging (PAI) prototype endoscopy system for ovarian tissue characterization. The overall diameter of the prototype endoscope is 5 mm which is suitable for insertion through a standard 5-12.5mm endoscopic laparoscopic port during minimally invasive surgery. It consists of a ball-lensed OCT sample arm probe, a multimode fiber having the output end polished at 45 degree angle so as to deliver the light perpendicularly for PAI, and a high frequency ultrasound transducer with 35MHz center frequency. System characterizations of OCT, US and PAI are presented. In addition, results obtained from ex vivo porcine and human ovarian tissues are presented. The optical absorption contrast provided by PAI, the high resolution subsurface morphology provided by OCT, and the deeper tissue structure imaged by US demonstrate the synergy of the combined endoscopy and the superior performance of this hybrid device over each modality alone in ovarian tissue characterization.

Self-separated PZT thick films with bulk-like piezoelectric and electromechanical properties.

Self-separated Pb(Zr(0.52)Ti(0.48))O(3) (PZT) films were processed by a hydrothermal deposition and a rapid thermal separation method, followed by a sol-gel filling and sintering process. The films possess excellent piezoelectric and electromechanical properties close to those of bulk material. The maximum remnant polarization is over 30 µC/cm(2) and the electromechanical coupling factor (k(t)) reaches as high as 0.52. The unique microstructure characteristics of the PZT films, such as their highly dense structure, columnar grains, well-connected grain boundaries, and well-dispersed nanopores, could all contribute to the enhanced piezoelectric and electromechanical properties.

In vivo label-free photoacoustic microscopy of cell nuclei by excitation of DNA and RNA.

Imaging of cell nuclei plays a critical role in cancer diagnosis and prognosis. To image noninvasively cell nuclei in vivo without staining, we developed UV photoacoustic microscopy (UV-PAM), in which 266 nm wavelength UV light excites unlabeled DNA and RNA in cell nuclei to produce photoacoustic waves. We applied UV-PAM to ex vivo imaging of cell nuclei in a mouse lip and a mouse small intestine and to in vivo imaging of the cell nuclei in the mouse skin. The UV-PAM images of unstained cell nuclei match the optical micrographs of the histologically stained cell nuclei. Given intrinsic optical contrast and high spatial resolution, in vivo label-free UV-PAM has potential for unique biological and clinical application.

Micro-electrocardiograms to study post-ventricular amputation of zebrafish heart.

The zebrafish (Danio rerio) is an emerging model for cardiovascular research. The zebrafish heart regenerates after 20% ventricular amputation. However, assessment of the physiological responses during heart regeneration has been hampered by the small size of the heart and the necessity of conducting experiments in an aqueous environment. We developed a methodology to monitor a real-time surface electrocardiogram (ECG) by the use of micro-electrodes, signal amplification, and a low pass-filter at a sampling rate of 1 kHz. Wavelet transform was used to further remove ambient noises. Rather than paralyzing the fish, we performed mild sedation by placing the fish in a water bath mixed with MS-222 (tricane methanesulfonate). We recorded distinct P waves for atrial contraction, QRS complexes for ventricular depolarization, and QT intervals for ventricular repolarization prior to, and 2 and 4 days post-amputation (dpa). Sedation reduced the mean fish heart rate from 149 +/- 18 to 90 +/- 17 beats/min. The PR and QRS intervals remained unchanged in response to ventricular apical amputation (n = 6, p > 0.05). Corrected QT intervals (QTc) were shortened 4 dpa (n = 6, p < 0.05). In a parallel study, histology revealed that apical thrombi were replaced with fibrin clots and collagen fibers. Atrial arrhythmia was noted in response to prolonged sedation. Unlike the human counterpart, ventricular tachycardia or fibrillation was not observed in response to ventricular amputation 2 and 4 dpa. Taken together, we demonstrated a minimally invasive methodology to monitor zebrafish heart function, electrical activities, and regeneration in real-time.