In vivo acoustic patterning of endothelial cells for tissue vascularization.

Strategies to fabricate microvascular networks that structurally and functionally mimic native microvessels are needed to address a host of clinical conditions associated with tissue ischemia. The objective of this work was to advance a novel ultrasound technology to fabricate complex, functional microvascular networks directly in vivo. Acoustic patterning utilizes forces within an ultrasound standing wave field (USWF) to organize cells or microparticles volumetrically into defined geometric assemblies. A dual-transducer system was developed to generate USWFs site-specifically in vivo through interference of two ultrasound fields. The system rapidly patterned injected cells or microparticles into parallel sheets within collagen hydrogels in vivo. Acoustic patterning of injected endothelial cells within flanks of immunodeficient mice gave rise to perfused microvessels within 7 days of patterning, whereas non-patterned cells did not survive. Thus, externally-applied ultrasound fields guided injected endothelial cells to self-assemble into perfused microvascular networks in vivo. These studies advance acoustic patterning towards in vivo tissue engineering by providing the first proof-of-concept demonstration that non-invasive, ultrasound-mediated cell patterning can be used to fabricate functional microvascular networks directly in vivo.

Phase holograms for the three-dimensional patterning of unconstrained microparticles.

Acoustic radiation forces can remotely manipulate particles. Forces from a standing wave field align microscale particles along the nodal or anti-nodal locations of the field to form three-dimensional (3D) patterns. These patterns can be used to form 3D microstructures for tissue engineering applications. However, standing wave generation requires more than one transducer or a reflector, which is challenging to implement in vivo. Here, a method is developed and validated to manipulate microspheres using a travelling wave from a single transducer. Diffraction theory and an iterative angular spectrum approach are employed to design phase holograms to shape the acoustic field. The field replicates a standing wave and aligns polyethylene microspheres in water, which are analogous to cells in vivo, at pressure nodes. Using Gor'kov potential to calculate the radiation forces on the microspheres, axial forces are minimized, and transverse forces are maximized to create stable particle patterns. Pressure fields from the phase holograms and resulting particle aggregation patterns match predictions with a feature similarity index > 0.92, where 1 is a perfect match. The resulting radiation forces are comparable to those produced from a standing wave, which suggests opportunities for in vivo implementation of cell patterning toward tissue engineering applications.

Time- and Dose-Dependent Effects of Pulsed Ultrasound on Dermal Repair in Diabetic Mice.

Chronic wounds, including diabetic, leg and pressure ulcers, impose a significant health care burden worldwide. Some evidence indicates that ultrasound can enhance soft tissue repair. However, therapeutic responses vary among individuals, thereby limiting clinical translation. Here, effects of pulsed ultrasound on dermal wound healing were assessed using a murine model of chronic, diabetic wounds. An ultrasound exposure system was developed to provide daily ultrasound exposures to full-thickness, excisional wounds in genetically diabetic mice. Wounds were exposed to 1 MHz ultrasound (2 ms pulse, 100 Hz pulse repetition frequency, 0-0.4 MPa) for 2 or 3 wk. Granulation tissue thickness and wound re-epithelialization increased as a function of increasing ultrasound pressure amplitude. At 2 wk after injury, significant increases in granulation tissue thickness and epithelial ingrowth were observed in response to 1 MHz pulsed ultrasound at 0.4 MPa. Wounds exposed to 0.4 MPa ultrasound for 3 wk were characterized by collagen-dense, revascularized granulation tissue with a fully restored, mature epithelium. Of note, only half of wounds exposed to 0.4 MPa ultrasound showed significant granulation tissue deposition after 2 wk of treatment. Thus, the db+/db+ mouse model may help to identify biological variables that influence individual responses to pulsed ultrasound and accelerate clinical translation.

Acoustic Fabrication of Collagen-Fibronectin Composite Gels Accelerates Microtissue Formation.

Ultrasound can influence biological systems through several distinct acoustic mechanisms that can be manipulated by varying reaction conditions and acoustic exposure parameters. We recently reported a new ultrasound-based fabrication technology that exploits the ability of ultrasound to generate localized mechanical forces and thermal effects to control collagen fiber microstructure non-invasively. Exposing solutions of type I collagen to ultrasound during the period of microfibril assembly produced changes in collagen fiber structure and alignment, and increased the biological activity of the resultant collagen hydrogels. In the extracellular matrix, interactions between fibronectin and collagen fibrils influence the biological activity of both proteins. Thus, in the present study, we examined how addition of fibronectin to collagen solutions prior to ultrasound exposure affects protein organization and the biological activity of the composite hydrogels. Results indicate that ultrasound can alter the distribution of fibronectin within 3D hydrogels via thermal and non-thermal mechanisms to produce composite hydrogels that support accelerated microtissue formation. The use of acoustic energy to drive changes in protein conformation to functionalize biomaterials has much potential as a unique, non-invasive technology for tissue engineering and regenerative medicine.

Using Acoustic Fields to Fabricate ECM-Based Biomaterials for Regenerative Medicine Applications.

Ultrasound is emerging as a promising tool for both characterizing and fabricating engineered biomaterials. Ultrasound-based technologies offer a diverse toolbox with outstanding capacity for optimization and customization within a variety of therapeutic contexts, including improved extracellular matrix-based materials for regenerative medicine applications. Non-invasive ultrasound fabrication tools include the use of thermal and mechanical effects of acoustic waves to modify the structure and function of extracellular matrix scaffolds both directly, and indirectly via biochemical and cellular mediators. Materials derived from components of native extracellular matrix are an essential component of engineered biomaterials designed to stimulate cell and tissue functions and repair or replace injured tissues. Thus, continued investigations into biological and acoustic mechanisms by which ultrasound can be used to manipulate extracellular matrix components within three-dimensional hydrogels hold much potential to enable the production of improved biomaterials for clinical and research applications.

Diagnostic Ultrasound Safety Review for Point-of-Care Ultrasound Practitioners.

Potential ultrasound exposure safety issues are reviewed, with guidance for prudent use of point-of-care ultrasound (POCUS). Safety assurance begins with the training of POCUS practitioners in the generation and interpretation of diagnostically valid and clinically relevant images. Sonographers themselves should minimize patient exposure in accordance with the as-low-as-reasonably-achievable principle, particularly for the safety of the eye, lung, and fetus. This practice entails the reduction of output indices or the exposure duration, consistent with the acquisition of diagnostically definitive images. Informed adoption of POCUS worldwide promises a reduction of ionizing radiation risks, enhanced cost-effectiveness, and prompt diagnoses for optimal patient care.

Non-invasive acoustic fabrication methods to enhance collagen hydrogel bioactivity.

Much attention has focused recently on utilizing components of the extracellular matrix (ECM) as natural building blocks for a variety of tissue engineering applications and regenerative medicine therapies. Consequently, new fabrication methods are being sought to enable molecular control over the structural characteristics of ECM molecules in order to improve their biological function. Exposing soluble collagen to acoustic forces associated with ultrasound propagation produces localized variations in collagen microfiber organization that in turn, promote cell behaviors essential for tissue regeneration, including cell migration and matrix remodeling. In the present study, mechanisms by which ultrasound interacts with polymerizing collagen to produce functional changes in collagen microstructure were investigated. The rate of collagen polymerization was manipulated by adjusting the pH of collagen solutions and the temperature at which gels were polymerized. Results demonstrate that the phase transition of type I collagen from fluid to gel triggered a simultaneous increase in acoustic absorption. This phase transition of collagen involves the lateral growth of early-stage collagen microfibrils and importantly, corresponded to a defined period of time during which exposure to ultrasound introduced both structural and functional changes to the resultant collagen hydrogels. Together, these experiments isolated a critical window in the collagen fiber assembly process during which mechanical forces associated with ultrasound propagation are effective in producing structural changes that underlie the ability of acoustically-modified collagen hydrogels to stimulate cell migration. These results demonstrate that changes in material properties associated with collagen polymerization are a fundamental component of the mechanism by which acoustic forces modify collagen biomaterials to enhance biological function.

Ultrasound patterning technologies for studying vascular morphogenesis in 3D.

Investigations in this report demonstrate the versatility of ultrasound-based patterning and imaging technologies for studying determinants of vascular morphogenesis in 3D environments. Forces associated with ultrasound standing wave fields (USWFs) were employed to non-invasively and volumetrically pattern endothelial cells within 3D collagen hydrogels. Patterned hydrogels were composed of parallel bands of endothelial cells located at nodal regions of the USWF and spaced at intervals equal to one half wavelength of the incident sound field. Acoustic parameters were adjusted to vary the spatial dimensions of the endothelial bands, and effects on microvessel morphogenesis were analyzed. High-frequency ultrasound imaging techniques were used to image and quantify the spacing, width and density of initial planar cell bands. Analysis of resultant microvessel networks showed that vessel width, orientation, density and branching activity were strongly influenced by the initial 3D organization of planar bands and, hence, could be controlled by acoustic parameters used for patterning. In summary, integration of USWF-patterning and high-frequency ultrasound imaging tools enabled fabrication of vascular constructs with defined microvessel size and orientation, providing insight into how spatial cues in 3D influence vascular morphogenesis.

Biological Effects of Low-Frequency Shear Strain: Physical Descriptors.

Biological effects of megahertz-frequency diagnostic ultrasound are thoroughly monitored by professional societies throughout the world. A corresponding, thorough, quantitative evaluation of the archival literature on the biological effects of low-frequency vibration is needed. Biological effects, of course, are related directly to what those exposures do physically to the tissue-specifically, to the shear strains that those sources produce in the tissues. Instead of the simple compressional strains produced by diagnostic ultrasound, realistic sources of low-frequency vibration produce both fast (∼1,500 m/s) and slow (1-10 m/s) waves, each of which may have longitudinal and transverse shear components. Part 1 of this series illustrates the resulting strains, starting with those produced by longitudinally and transversely oscillating planes, through monopole and dipole sources of fast waves and, finally, to the case of a sphere moving in translation-the simplest model of the fields produced by realistic sources.

Quantitative Ultrasound for Nondestructive Characterization of Engineered Tissues and Biomaterials.

Non-invasive, non-destructive technologies for imaging and quantitatively monitoring the development of artificial tissues are critical for the advancement of tissue engineering. Current standard techniques for evaluating engineered tissues, including histology, biochemical assays and mechanical testing, are destructive approaches. Ultrasound is emerging as a valuable tool for imaging and quantitatively monitoring the properties of engineered tissues and biomaterials longitudinally during fabrication and post-implantation. Ultrasound techniques are rapid, non-invasive, non-destructive and can be easily integrated into sterile environments necessary for tissue engineering. Furthermore, high-frequency quantitative ultrasound techniques can enable volumetric characterization of the structural, biological, and mechanical properties of engineered tissues during fabrication and post-implantation. This review provides an overview of ultrasound imaging, quantitative ultrasound techniques, and elastography, with representative examples of applications of these ultrasound-based techniques to the field of tissue engineering.

Comparison of Thermal Safety Practice Guidelines for Diagnostic Ultrasound Exposures.

This article examines the historical evolution of various practice guidelines designed to minimize the possibility of thermal injury during a diagnostic ultrasound examination, including those published by the American Institute of Ultrasound in Medicine, British Medical Ultrasound Society and Health Canada. The guidelines for prenatal/neonatal examinations are in general agreement, but significant differences were found for postnatal exposures. We propose sets of thermal index versus exposure time for these examination categories below which there is reasonable assurance that an examination can be conducted without risk of producing an adverse thermal effect under any scanning conditions. If it is necessary to exceed these guidelines, the occurrence of an adverse thermal event is still unlikely in most situations because of mitigating factors such as transducer movement and perfusion, but the general principle of "as low as reasonably achievable" should be followed. Some limitations of the biological effects studies underpinning the guidelines also are discussed briefly.

Scholte wave generation during single tracking location shear wave elasticity imaging of engineered tissues.

The physical environment of engineered tissues can influence cellular functions that are important for tissue regeneration. Thus, there is a critical need for noninvasive technologies capable of monitoring mechanical properties of engineered tissues during fabrication and development. This work investigates the feasibility of using single tracking location shear wave elasticity imaging (STL-SWEI) for quantifying the shear moduli of tissue-mimicking phantoms and engineered tissues in tissue engineering environments. Scholte surface waves were observed when STL-SWEI was performed through a fluid standoff, and confounded shear moduli estimates leading to an underestimation of moduli in regions near the fluid-tissue interface.

Ultrasound technologies for biomaterials fabrication and imaging.

Ultrasound is emerging as a powerful tool for developing biomaterials for regenerative medicine. Ultrasound technologies are finding wide-ranging, innovative applications for controlling the fabrication of bioengineered scaffolds, as well as for imaging and quantitatively monitoring the properties of engineered constructs both during fabrication processes and post-implantation. This review provides an overview of the biomedical applications of ultrasound for imaging and therapy, a tutorial of the physical mechanisms through which ultrasound can interact with biomaterials, and examples of how ultrasound technologies are being developed and applied for biomaterials fabrication processes, non-invasive imaging, and quantitative characterization of bioengineered scaffolds in vitro and in vivo.

Noninvasive Quantitative Imaging of Collagen Microstructure in Three-Dimensional Hydrogels Using High-Frequency Ultrasound.

Collagen I is widely used as a natural component of biomaterials for both tissue engineering and regenerative medicine applications. The physical and biological properties of fibrillar collagens are strongly tied to variations in collagen fiber microstructure. The goal of this study was to develop the use of high-frequency quantitative ultrasound to assess collagen microstructure within three-dimensional (3D) hydrogels noninvasively and nondestructively. The integrated backscatter coefficient (IBC) was employed as a quantitative ultrasound parameter to detect, image, and quantify spatial variations in collagen fiber density and diameter. Collagen fiber microstructure was varied by fabricating hydrogels with different collagen concentrations or polymerization temperatures. IBC values were computed from measurements of the backscattered radio-frequency ultrasound signals collected using a single-element transducer (38-MHz center frequency, 13-47 MHz bandwidth). The IBC increased linearly with increasing collagen concentration and decreasing polymerization temperature. Parametric 3D images of the IBC were generated to visualize and quantify regional variations in collagen microstructure throughout the volume of hydrogels fabricated in standard tissue culture plates. IBC parametric images of corresponding cell-embedded collagen gels showed cell accumulation within regions having elevated collagen IBC values. The capability of this ultrasound technique to noninvasively detect and quantify spatial differences in collagen microstructure offers a valuable tool to monitor the structural properties of collagen scaffolds during fabrication, to detect functional differences in collagen microstructure, and to guide fundamental research on the interactions of cells and collagen matrices.

Estimating cell concentration in three-dimensional engineered tissues using high frequency quantitative ultrasound.

Histology and biochemical assays are standard techniques for estimating cell concentration in engineered tissues. However, these techniques are destructive and cannot be used for longitudinal monitoring of engineered tissues during fabrication processes. The goal of this study was to develop high-frequency quantitative ultrasound techniques to nondestructively estimate cell concentration in three-dimensional (3-D) engineered tissue constructs. High-frequency ultrasound backscatter measurements were obtained from cell-embedded, 3-D agarose hydrogels. Two broadband single-element transducers (center frequencies of 30 and 38 MHz) were employed over the frequency range of 13-47 MHz. Agarose gels with cell concentrations ranging from 1 × 10(4) to 1 × 10(6) cells mL(-1) were investigated. The integrated backscatter coefficient (IBC), a quantitative ultrasound spectral parameter, was calculated and used to estimate cell concentration. Accuracy and precision of this technique were analyzed by calculating the percent error and coefficient of variation of cell concentration estimates. The IBC increased linearly with increasing cell concentration. Axial and lateral dimensions of regions of interest that resulted in errors of less than 20% were determined. Images of cell concentration estimates were employed to visualize quantitatively regional differences in cell concentrations. This ultrasound technique provides the capability to rapidly quantify cell concentration within 3-D tissue constructs noninvasively and nondestructively.

Regional fibronectin and collagen fibril co-assembly directs cell proliferation and microtissue morphology.

The extracellular matrix protein, fibronectin stimulates cells to self-assemble into three-dimensional multicellular structures by a mechanism that requires the cell-dependent conversion of soluble fibronectin molecules into insoluble fibrils. Fibronectin also binds to collagen type I and mediates the co-assembly of collagen fibrils into the extracellular matrix. Here, the role of collagen-fibronectin binding in fibronectin-induced cellular self-assembly was investigated using fibronectin-null fibroblasts in an in vitro model of tissue formation. High resolution, two-photon immunofluorescence microscopy was combined with second harmonic generation imaging to examine spatial and temporal relationships among fibronectin and collagen fibrils, actin organization, cell proliferation, and microtissue morphology. Time course studies coupled with simultaneous 4-channel multiphoton imaging identified regional differences in fibronectin fibril conformation, collagen fibril remodeling, actin organization, and cell proliferation during three-dimensional cellular self-assembly. Regional differences in cell proliferation and fibronectin structure were dependent on both soluble fibronectin concentration and fibronectin-collagen interactions. Fibronectin-collagen binding was not necessary for either fibronectin matrix formation or intercellular cohesion. However, inhibiting fibronectin binding to collagen reduced collagen fibril remodeling, decreased fibronectin fibril extension, blocked fibronectin-induced cell proliferation, and altered microtissue morphology. Furthermore, continual fibronectin-collagen binding was necessary to maintain both cell proliferation and microtissue morphology. Collectively, these data suggest that the complex changes in extracellular matrix and cytoskeletal remodeling that mediate tissue assembly are driven, in part, by regional variations in cell-mediated fibronectin-collagen co-assembly.

Spatial patterning of endothelial cells and vascular network formation using ultrasound standing wave fields.

The spatial organization of cells is essential for proper tissue assembly and organ function. Thus, successful engineering of complex tissues and organs requires methods to control cell organization in three dimensions. In particular, technologies that facilitate endothelial cell alignment and vascular network formation in three-dimensional tissue constructs would provide a means to supply essential oxygen and nutrients to newly forming tissue. Acoustic radiation forces associated with ultrasound standing wave fields can rapidly and non-invasively organize cells into distinct multicellular planar bands within three-dimensional collagen gels. Results presented herein demonstrate that the spatial pattern of endothelial cells within three-dimensional collagen gels can be controlled by design of acoustic parameters of the sound field. Different ultrasound standing wave field exposure parameters were used to organize endothelial cells into either loosely aggregated or densely packed planar bands. The rate of vessel formation and the morphology of the resulting endothelial cell networks were affected by the initial density of the ultrasound-induced planar bands of cells. Ultrasound standing wave fields provide a rapid, non-invasive approach to pattern cells in three-dimensions and direct vascular network formation and morphology within engineered tissue constructs.

Controlling collagen fiber microstructure in three-dimensional hydrogels using ultrasound.

Type I collagen is the primary fibrillar component of the extracellular matrix, and functional properties of collagen arise from variations in fiber structure. This study investigated the ability of ultrasound to control collagen microstructure during hydrogel fabrication. Under appropriate conditions, ultrasound exposure of type I collagen during polymerization altered fiber microstructure. Scanning electron microscopy and second-harmonic generation microscopy revealed decreased collagen fiber diameters in response to ultrasound compared to sham-exposed samples. Results of mechanistic investigations were consistent with a thermal mechanism for the effects of ultrasound on collagen fiber structure. To control collagen microstructure site-specifically, a high frequency, 8.3-MHz, ultrasound beam was directed within the center of a large collagen sample producing dense networks of short, thin collagen fibrils within the central core of the gel and longer, thicker fibers outside the beam area. Fibroblasts seeded onto these gels migrated rapidly into small, circularly arranged aggregates only within the beam area, and clustered fibroblasts remodeled the central, ultrasound-exposed collagen fibrils into dense sheets. These investigations demonstrate the capability of ultrasound to spatially pattern various collagen microstructures within an engineered tissue noninvasively, thus enhancing the level of complexity of extracellular matrix microenvironments and cellular functions achievable within three-dimensional engineered tissues.

Fibronectin matrix mimetics promote full-thickness wound repair in diabetic mice.

During tissue repair, fibronectin is converted from a soluble, inactive form into biologically active extracellular matrix (ECM) fibrils through a cell-dependent process. ECM fibronectin promotes numerous cell processes that are critical to tissue repair and regulates the assembly of other proteins into the matrix. Nonhealing wounds show reduced levels of ECM fibronectin. To functionally mimic ECM fibronectin, a series of fibronectin matrix mimetics was developed by directly coupling the matricryptic, heparin-binding fragment of the first type III repeat of fibronectin (FNIII1H) to various sequences from the integrin-binding domain (FNIII8-10). The recombinant proteins were produced as glutathione-S-transferase (GST)-tagged fusion proteins for ease of production and purification. Full-thickness, excisional wounds were produced in genetically diabetic mice, and fibronectin matrix mimetics were applied directly to the wounds. A significant enhancement of wound closure was observed by day 9 in response to GST/III1H,8-10 versus GST-treated controls (73.9%±4.1% vs. 58.1%±4.7% closure, respectively). Two weeks after injury, fibronectin matrix mimetic-treated wounds had developed a multi-layered epithelium that completely covered the wound space. Furthermore, significant increases in granulation tissue thickness were observed in response to treatment with GST/III1H,8-10 (4.05±0.93-fold), GST/III1H,8,10 (2.91±0.49-fold), or GST/III1H,8(RGD) (3.55±0.59-fold) compared with GST controls, and was accompanied by dense collagen deposition, the presence of myofibroblasts, and functional vasculature. Thus, the recombinant fibronectin matrix analogs normalized the impairment in repair observed in this chronic wound model and may provide a new approach to accelerate the healing of diabetic wounds.

Natural frequencies of two bubbles in a compliant tube: analytical, simulation, and experimental results.

Motivated by various clinical applications of ultrasound contrast agents within blood vessels, the natural frequencies of two bubbles in a compliant tube are studied analytically, numerically, and experimentally. A lumped parameter model for a five degree of freedom system was developed, accounting for the compliance of the tube and coupled response of the two bubbles. The results were compared to those produced by two different simulation methods: (1) an axisymmetric coupled boundary element and finite element code previously used to investigate the response of a single bubble in a compliant tube and (2) finite element models developed in comsol Multiphysics. For the simplified case of two bubbles in a rigid tube, the lumped parameter model predicts two frequencies for in- and out-of-phase oscillations, in good agreement with both numerical simulation and experimental results. For two bubbles in a compliant tube, the lumped parameter model predicts four nonzero frequencies, each asymptotically converging to expected values in the rigid and compliant limits of the tube material.