32 in Year 2013

Phase shift liquid perfluorocarbon (PFC) droplets vaporizable by ultrasound into echogenicmicrobubble above a threshold pressure, termed acoustic droplet vaporization (ADV), are usedfor therapeutic and diagnostic applications. This study systematically investigated the effect ofexcitation frequency (2.25, 10, and 15 MHz) on the ADV and inertial cavitation (IC) thresholds oflipid-coated PFC droplets of three different liquid cores—perfluoropentane (PFP), perfluorohexane(PFH), and perfluorooctyl bromide (PFOB)—and of two different sizes—average diameters smallerthan 3lm and larger than 10lm—in a tubeless setup. This study found that the ADV thresholdincreases with frequency for the lowest boiling point liquid, PFP, for both large and small sizedroplets. For higher boiling point liquids, PFH and PFOB, this study did not detect vaporization forsmall size droplets at the excitation levels (maximum 4 MPa peak negative) studied here. The largePFOB droplets experienced ADV only at the highest excitation frequency 15 MHz. For large PFHdroplets, ADV threshold decreases with frequency that could possibly be due to the superharmonicfocusing being a significant effect at larger sizes and the higher excitation pressures. ADV thresh-olds at all the frequencies studied here occurred at lower rarefactional pressures than IC thresholdsindicating that phase transition precedes inertial cavitation.

The shear-induced collective diffusivity down a concentration gradient in a viscous
emulsion is computed using direct numerical simulation. A layer of randomly packed
drops subjected to a shear flow, shows the layer width to increase with the 1=3
power of time, consistent with a semi-dilute theory that assumes a diffusivity linear
with concentration. This characteristic scaling and the underlying theory are used
to compute the collective diffusivity coefficient. This is the first ever computation
of this quantity for a system of deformable particles using fully resolved numerical
simulation. The results match very well with previous experimental observations.
The coefficient of collective diffusivity varies non-monotonically with the capillary
number, due to the competing effects of increasing deformation and drop orientation.
A phenomenological correlation for the collective diffusivity coefficient as a function
of capillary number is presented. We also apply an alternative approach to compute
collective diffusivity, developed originally for a statistically homogeneous rigid sphere
suspension – computing the dynamic structure factor from the simulated droplet
positions and examining its time variation at small wavenumber. We show that
the results from this alternative approach qualitatively agree with our computation
of collective diffusivity including the prediction of the non-monotonic variation of
diffusivity with the capillary number.

Phase shift droplets vaporizable by acoustic stimulation offer the advantages of producing micro-bubbles as contrast agentsin situas well as higher stability and the possibility of achieving smallersizes. Here, the acoustic droplet vaporization (ADV) threshold of a suspension of droplets with aperfluoropentane (PFP) core (diameter 400–3000 nm) is acoustically measured as a function of theexcitation frequency in a tubeless setup at room temperature. The changes in scattered responses—fundamental, sub-, and second harmonic—are investigated, a quantitative criterion is used to deter-mine the ADV phenomenon, and findings are discussed. The average threshold obtained using threedifferent scattered components increases with frequency—1.0560.28 MPa at 2.25 MHz,1.8960.57 MPa at 5 MHz, and 2.3460.014 MPa at 10 MHz. The scattered response from vapor-ized droplets was also found to qualitatively match with that from an independently prepared lipid-coated microbubble suspension in magnitude as well as trends above the determined ADV thresh-old value.

Hypoxia in solid tumors facilitates the progres-sion of the disease, develops resistance to chemo and radiotherapy, and contributes to relapse. Due to the lack of tumor penetration, most of the reported drug carriers are unable to reach the hypoxic niches of the solid tumors. We have developed tissue-penetrating, hypoxia-responsive echogenic polymersomes to deliver anti cancer drugs to solid tumors. The polymersomes are composed of a hy-poxia-responsive azobenzene conjugated and a tissue penetrating peptide functionalized polylactic acid-polyethylene glycol polymer. The drug-encapsulated, hypoxia-responsive polymersomes substantially decreased the viability of pancreatic cancer cells in spheroidal cultures. Under normoxic conditions, polymersomes were echogenic at diagnostic ultrasound frequencies but lose the echogenicity under hypoxia. In vivo imaging studies with xenograft mouse model further confirmed the ability of the polymersomes to target, penetrate, and deliver the encapsulated contents in hypoxic pancreatic tumor tissues.

The acoustic and mechanical properties of 3D-printed porous poly-(ethylene glycol)-diacrylate(PEGDA)hydrogel scaffolds were investigated using an ultrasound pulse echo technique on differentscaffold microstructures(solid, hexagonal and square pores). Acoustic parameters such as speed ofsound, acoustic impedance and attenuation coefficient as well as physical parameters such as the porestructure, effective density and elastic moduli were determined. The results show that microstructure(porosity and pore geometry)plays a crucial role in defining properties of 3D-printed scaffolds,achieving the highest attenuation for the scaffold with hexagonal pores and showing a decrease insound speed and elastic moduli with increasing porosity. The properties were also found to be similarto those of soft tissues, making PEGDA scaffolds a suitable candidate for tissue engineeringapplications. To evaluate their cellular performance, adhesion and proliferation of humanmesenchymal stem cells(hMSCs)in these scaffolds were investigated. The porous scaffolds performedbetter than the solid one, recording the highest cell attachment and growth for the scaffold with thesquare pores.

Chemotherapeutic agents for treating cancers show considerable sideeffects, toxicity, and drug resistance. To mitigate the problems, we designed nucleus-targeted, echogenic, stimuli-responsive polymeric vesicles (polymersomes) to transport andsubsequently release the encapsulated anticancer drugs within the nuclei of pancreaticcancer cells. We synthesized an alkyne-dexamethasone derivative and conjugated it to N3−polyethylene glycol (PEG)−polylactic acid (PLA) copolymer employing the Cu2+catalyzed“Click”reaction. We prepared polymersomes from the dexamethasone−PEG−PLA conjugate along with a synthesized stimuli-responsive polymer PEG−S−S−PLA. Thedexamethasone group dilates the nuclear pore complexes and transports the vesicles to thenuclei. We designed the polymersomes to release the encapsulated drugs in the presence ofa high concentration of reducing agents in the nuclei of pancreatic cancer cells. Weobserved that the nucleus-targeted, stimuli-responsive polymersomes released 70% ofencapsulated contents in the nucleus-mimicking environment in 80 min. We encapsulatedthe cancer stemness inhibitor BBI608 in the vesicles and observed that the BBI608encapsulated polymersomes reduced the viability of the BxPC3 cells to 43% in three-dimensional spheroid cultures. Thepolymersomes were prepared following a special protocol so that they scatter ultrasound, allowing imaging by a medicalultrasound scanner. Therefore, these echogenic, targeted, stimuli-responsive, and drug-encapsulated polymersomes have thepotential for trackable, targeted carrier of chemotherapeutic drugs to cancer cell nuclei.

Lipid-coated microbubbles, clinically approved as contrast enhancing agents for ultrasound imaging, are investigated for the first time for their possible applications in bone tissue engineering. Effects of microbubbles (average diameter 1.1 μm) coated by a mixture of lipids (1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000], and 1,2-dipalmitoyl-3-trimethylmmonium-propane) in the presence of low intensity pulsed ultrasound (LIPUS) on human mesenchymal stem cells seeded on 3D printed poly(lactic acid) porous scaffolds are investigated. LIPUS stimulation (30 mW cm−2, 1.5 MHz, 20% duty cycle) for 3 min a day with 0.5% v/v microbubbles results in a significant increase in proliferation (up to 19.3%) when compared to control after 1, 3, and 5 d. A 3-week osteogenic differentiation study shows a significant increase in total protein content (up to 27.5%), calcium deposition (up to 4.3%), and alkaline phosphatase activity (up to 43.1%) initiated by LIPUS with and without the presence of microbubbles. The microbubbles are found to remain stable during exposure, and their sustained oscillations demonstrably help focus the LIPUS energy toward enhanced cellular response. Integrating LIPUS and microbubbles promises to be a novel and effective strategy for bone tissue engineering and regeneration therapies.

Four dimensional (4D) printing is an emerging technology with great capacity for fabricating complex,stimuli-responsive 3D structures, providing great potential for tissue and organ engineering applica-tions. Although the 4D concept wasfirst highlighted in 2013, extensive research has rapidly developed,along with more-in-depth understanding and assertions regarding the definition of 4D. In this review,we begin by establishing the criteria of 4D printing, followed by an extensive summary of state-of-the-art technological advances in thefield. Both transformation-preprogrammed 4D printing and 4Dprinting of shape memory polymers are intensively surveyed. Afterwards we will explore and discussthe applications of 4D printing in tissue and organ regeneration, such as developing synthetic tissuesand implantable scaffolds, as well as future perspectives and conclusions.

Echogenic liposomes (ELIPs) are an excellent candidate for ultrasound activated therapeutics andimaging. Although multiple experiments have established their echogenicity, the underlying mech-anism has remained unknown. However, freeze-drying in the presence of mannitol during ELIPpreparation has proved critical to ensuring echogenicity. Here, the role of this key component in thepreparation protocol was investigated by measuring scattering from freshly prepared freeze-driedaqueous solution of mannitol—and a number of other excipients commonly used in lyophiliza-tion—directly dispersed in water without any lipids in the experiment. Mannitol, meso-erythritol,glycine, and glucose that form a highly porous crystalline phase upon freeze-drying generated bub-bles resulting in strong echoes during their dissolution. On the other hand, sucrose, trehalose, andxylitol, which become glassy while freeze-dried, did not. Freeze-dried mannitol and other crystal-line substances, if thawed before being introduced into the scattering volume, did not produce echo-genicity, as they lost their crystallinity in the thawed state. The echogenicity disappeared in adegassed environment. Higher amounts of sugar in the original aqueous solution before freeze-drying resulted in higher echogenicity because of the stronger supersaturation and crystallinity. Thebubbles created by the freeze-dried mannitol in the ELIP formulation play a critical role in makingELIPs echogenic.

Polymersomes are a class of artificial vesicles prepared from amphiphilic polymers. Like lipid vesicles(liposomes), they too can encapsulate hydrophilic and hydrophobic drug molecules in the aqueous core and thehydrophobic bilayer respectively, but are more stable than liposomes. Although echogenic liposomes have beenwidely investigated for simultaneous ultrasound imaging and controlled drug delivery, the potential of thepolymersomes remains unexplored. We prepared two different echogenic polymersomes from the amphiphilic co-polymers polyethylene glycol–poly-DL-lactic acid (PEG-PLA) and polyethylene glycol–poly-L-lactic acid (PEG-PLLA), incorporating multiple freeze-dry cycles in the synthesis protocol to ensure their echogenicity. We investigatedacoustic behavior with potential applications in biomedical imaging. We characterized the polymeric vesicles acous-tically with three different excitation frequencies of 2.25, 5 and 10 MHz at 500 kPa. The polymersomes exhibitedstrong echogenicity at all three excitation frequencies (about 50- and 25-dB enhancements in fundamental andsubharmonic, respectively, at 5-MHz excitation from 20g/mL polymers in solution). Unlike echogenic lipo-somes, they emitted strong subharmonic responses. The scattering results indicated their potential as contrastagents, which was also confirmed by clinical ultrasound imaging.

The effects of low intensity pulsed ultrasound (LIPUS) on proliferation andchondrogenic differentiation of human mesenchymal stem cells (hMSCs) seeded on3D printed poly-(ethylene glycol)-diacrylate (PEG-DA) scaffolds with varying poregeometries (square and hexagonal channels) were investigated. The scaffold withsquare pores resulted in higher hMSC growth and chondrogenic differentiation than asolid or a hexagonally porous scaffold. The optimal LIPUS parameters at 1.5 MHz werefound to be 100 mW/cm2and 20% duty cycle. LIPUS stimulation increasedproliferation by up to 60% after 24 hr. For chondrogenesis, we evaluated key cartilagebiomarkers abundant in cartilage tissue; glycosaminoglycan (GAG), type II collagen andtotal collagen. LIPUS stimulation enhanced GAG synthesis up to 16% and 11% forscaffoldswithsquareandhexagonalpatterns,respectively,after2weeks.Additionally,type II collagen production increased by 60% and 40% for the same patterns,respectively under LIPUS stimulation after 3 weeks. These results suggest that LIPUSstimulation, which has already been approved by FDA for treatment of bone fracture,could be a highly efficient tool for tissue engineering in combination with 3D printingand hMSCs to regenerate damaged cartilage tissues.

In this Technical Note, we determine the interfacial rheological parameters of the encapsulation of thecontrast agent Targestar P using ultrasound attenuation. The characteristic parameters are obtained according totwo interfacial rheological models. The properties—surface dilatational elasticity (0.09 ± 0.01 N/m) and surfacedilatational viscosity (8 ± 0.1E–9 N$s/m)—are found to be of similar magnitude for both models. Contrast micro-bubbles experience different ambient pressure in different organs. We also measure these parameters as functionsof ambient pressure using attenuation measured at different overpressures (0, 100 and 200 mm Hg). For each valueof ambient hydrostatic pressure, we determine the rheological properties, accounting for changes in the size dis-tribution caused by the pressure change. We discuss different models of size distribution change under overpres-sure: pure adiabatic compression or gas exchange with surrounding medium. The dilatational surface elasticityand viscosity are found to increase with increasing ambient pressure.

The shear rheology of an emulsion of viscous drops in the presence of finite inertiais investigated using direct numerical simulation. In the absence of inertia, emulsionsdisplay a non-Newtonian rheology with positive first and negative second normalstress differences. However, recently it was discovered that a small amount ofdrop-level inertia alters their signs – the first normal stress difference becomesnegative and the second one becomes positive, each in a small range of capillarynumbers (Li & Sarkar,J. Rheol., vol. 49, 2005, pp. 1377–1394). Sign reversal wasshown numerically and analytically, but only in the limit of a dilute emulsion wheredrop–drop interactions were neglected. Here, we compute the rheology of a density-and viscosity-matched emulsion, accounting for the interactions in the volume fractionrange of 5 %–27 % and Reynolds number range of 0.1–10. The computed rheologicalproperties (effective shear viscosity and first and second normal stress differences) inthe Stokes limit match well with previous theoretical (Choi–Schowalter in the dilutelimit) and simulated results (for concentrated systems) using the boundary elementmethod. The two distinct components of the rheology arising from the interfacialstresses at the drop surface and the perturbative Reynolds stresses are investigated asfunctions of the drop Reynolds number, capillary number and volume fraction. Thesign change is caused by the increasing drop inclination in the presence of inertia,which in turn directly affects the interfacial stresses. Increase of the volume fractionor capillary number increases the critical Reynolds number for sign reversals due toenhanced alignment of the drops with the flow directions. The effect of increasingthe volume fraction on the rheology is explained by relating it to interactions andspecifically to the contact pair-distribution function computed from the simulation.The excess stresses are seen to show an approximately linear behaviour with theReynolds number in the range of 0.1–5, while with the capillary number and volumefraction, the variation is weakly quadratic.

3D printing and ultrasound techniques are showing great promise in the evolution of human musculoskeletal tissue repair and regeneration medicine. The uniqueness of the present study was to combine low intensity pulsed ultrasound (LIPUS) and advanced 3D printing techniques to synergistically improve growth and osteogenic differentiation of human mesenchymal stem cells (MSC). Specifically, polyethylene glycol diacrylate bioinks containing cell adhesive Arginine-Glycine-Aspartic acid-Serene (RGDS) peptide and/or nanocrystalline hydroxyapatite (nHA) were used to fabricate 3D scaffolds with different geometric patterns via novel table-top stereolithography 3D printer. The resultant scaffolds provide a highly porous and interconnected 3D environment to support cell proliferation. Scaffolds with small square pores were determined to be the optimal geometric pattern for MSC attachment and growth. The optimal LIPUS working parameters were determined to be 1.5 MHz, 20% duty cycle with 150 mW/cm2 intensity. Results demonstrated that RGDS peptide and nHA containing 3D printed scaffolds under LIPUS treatment can greatly promote MSC proliferation, alkaline phosphatase activity, calcium deposition and total protein content. These results illustrate the effectiveness of the combination of LIPUS and biomimetic 3D printing scaffolds as a valuable combinatorial tool for improved MSC function, thus make them promising for future clinical and various regenerative medicine application.

Lipid-coated microbubbles are used to enhance ultrasound imaging and drug delivery. Here we apply these microbubbles along with low intensity pulsed ultrasound (LIPUS) for the first time to enhance proliferation and chondrogenic differentiation of human mesenchymal stem cells (hMSCs) in a 3D printed poly-(ethylene glycol)-diacrylate (PEG-DA) hydrogel scaffold. The hMSC proliferation increased up to 40% after 5 days of culture in the presence of 0.5% (v/v) microbubbles and LIPUS in contrast to 18% with LIPUS alone. We systematically varied the acoustic excitation parameters—excitation intensity, frequency and duty cycle—to find 30 mW/cm2, 1.5 MHz and 20% duty cycle to be optimal for hMSC proliferation. A 3-week chondrogenic differentiation results demonstrated that combining LIPUS with microbubbles enhanced glycosaminoglycan (GAG) production by 17% (5% with LIPUS alone), and type II collagen production by 78% (44% by LIPUS alone). Therefore, integrating LIPUS and microbubbles appears to be a promising strategy for enhanced hMSC growth and chondrogenic differentiation, which are critical components for cartilage regeneration. The results offer possibilities of novel applications of microbubbles, already clinically approved for contrast enhanced ultrasound imaging, in tissue engineering.

Hydrodynamic interactions between a pair of capsules in simple shear are numerically investigated using afront-tracking finite difference method. The membrane of the capsule is modeled using different hyperelasticconstitutive relations. We also compare the pair interactions between drops to those between capsules. Anincreased viscosity ratio leads to a reduced net cross-stream separation between capsules as well as drops aftercollision. At low viscosity ratios, for the same capillary number drop-pairs show higher cross-stream separationthan those for capsule-pairs, while substantially large viscosity ratios result in almost the same value for bothcases. We investigate pair-collisions between two heterogeneous capsules C1and C2with two different capillarynumbers. The maximum deformation of C1was seen to increase with increasing stiffness (decreasing capillarynumber) of C2, even though the stiffness of C1was kept fixed. The findings are similar for a drop-pair, however,with a smaller maximum deformation for the same combinations of capillary numbers. The final cross-streamdrift of the trajectory of C1decreases with the increasing stiffness of C2, but the relative trajectory betweenthe capsules remains unchanged. The maximum deformation and the cross-stream drift of the trajectory of C1are shown to approximately vary with power-law functions of the ratio of the capillary numbers of C1andC2. An analytical explanation of the dependence on the two capillary numbers is offered. Different membraneconstitutive laws result in similar deformation and drift in trajectory.

Ultrasound contrast microbubbles experience widely varying ambient blood pressure in differentorgans, which can also change due to diseases. Pressure change can alter the material properties ofthe encapsulation of these microbubbles. Here the characteristic rheological parameters of contrastagent Definity are determined by varying the ambient pressure (in a physiologically relevant range0–200 mm Hg). Four different interfacial rheological models are used to characterize the microbub-bles. Effects of gas diffusion under excess ambient pressure are investigated in detail accountingfor size decrease of contrast microbubbles. Definity contrast agent show a change in their interfacialdilatational viscosity (3.6108Ns/m at 0 mm Hg to 4.45108Ns/m at 200 mm Hg) and inter-facial dilatational elasticity (0.86 N/m at 0 mm Hg to 1.06 N/m at 200 mm Hg) with ambient pres-sure increase. The increase results from material consolidation, similar to such enhancement inbulk properties under pressure. The model that accounts for enhancement in material propertieswith increasing ambient pressure matches with experimentally measured subharmonic response asa function of ambient pressure, while assuming constant material parameters does not.

Broadband attenuation of ultrasound measured at different excitation pressures being differentraises a serious theoretical concern, because the underlying assumption of linear and independentpropagation of different frequency components nominally requires attenuation to be independent ofexcitation. Here, this issue is investigated by examining ultrasound attenuation through a monodis-perse lipid-coated microbubble suspension measured at four different acoustic excitation ampli-tudes. The attenuation data are used to determine interfacial rheological properties (surface tension,surface dilatational elasticity, and surface dilatational viscosity) of the encapsulation according tothree different models. Although different models result in similar rheological properties, attenua-tion measured at different excitation levels (4–110 kPa) leads to different values for them; the dila-tation elasticity (0.56 to 0.18 N/m) and viscosity (2.4108to 1.52108Ns/m) both decreasewith increasing pressure. Numerically simulating the scattered response, nonlinear energy transferbetween frequencies are shown to be negligible, thereby demonstrating the linearity in propagationand validating the attenuation analysis. There is a second concern to the characterization arisingfrom shell properties being dependent on excitation amplitude, which is not a proper constitutivevariable. It is resolved by arriving at a strain-dependent rheology for the encapsulation. The limita-tions of the underlying analysis are discussed.

Liposomes are representative lipid nanoparticles widelyused for delivering anticancer drugs, DNA fragments, or siRNA to cancercells. Upon targeting, various internal and external triggers have been usedto increase the rate for contents release from the liposomes. Among theinternal triggers, decreased pH within the cellular lysosomes has beensuccessfully used to enhance the rate for releasing contents. However,imparting pH sensitivity to liposomes requires the synthesis of specializedlipids with structures that are substantially modified at a reduced pH.Herein, we report an alternative strategy to render liposomes pH sensitiveby encapsulating a precursor which generates gas bubblesin situinresponse to acidic pH. The disturbance created by the escaping gasbubbles leads to the rapid release of the encapsulated contents from theliposomes. Atomic force microscopic studies indicate that the liposomalstructure is destroyed at a reduced pH. The gas bubbles also render theliposomes echogenic, allowing ultrasound imaging. To demonstrate the applicability of this strategy, we have successfully targeteddoxorubicin-encapsulated liposomes to the pancreatic ductal carcinoma cells that overexpress the folate receptor on the surface.In response to the decreased pH in the lysosomes, the encapsulated anticancer drug is efficiently released. Contents releasedfrom these liposomes are further enhanced by the application of continuous wave ultrasound (1 MHz), resulting in substantiallyreduced viability for the pancreatic cancer cells (14%).

The migration of a capsule enclosed by an elastic membrane in a wall-bounded linearshear is investigated using a front-tracking method. A detailed comparison with themigration of a viscous drop is presented varying the capillary number (in the caseof a capsule, the elastic capillary number) and the viscosity ratio. In both cases,the deformation breaks the flow reversal symmetry and makes them migrate awayfrom the wall. They quickly go through a transient evolution to eventually reach aquasi-steady state where the dynamics becomes independent of the initial positionand only depends on the wall distance. Previous analytical theories predicted thatfor a viscous drop, in the quasi-steady state, the migration and slip velocities scaleapproximately with the square of the inverse of the drop–wall separation, whereasthe drop deformation scales as the inverse cube of the separation. These power lawrelations are shown to hold for a capsule as well. The deformation and inclinationangle of the capsule and the drop at the same wall separation show a crossoverin their variation with the capillary number: the capsule shows a steeper variationthan that of the drop for smaller capillary numbers and slower variation than thedrop for larger capillary numbers. Using the Green’s function of Stokes flow, asemi-analytic theory is presented to show that the far-field stresslet that causes themigration has two distinct contributions from the interfacial stresses and the viscosityratio, with competing effects between the two defining the dynamics. It predicts thescaling of the migration velocity with the capsule–wall separation, however, matchingwith the simulated result very well only away from the wall. A phenomenologicalcorrelation for the migration velocity as a function of elastic capillary number, walldistance and viscosity ratio is developed using the simulation results. The effects ofdifferent membrane hyperelastic constitutive equations – neo-Hookean, Evans–Skalak,and Skalak – are briefly investigated to show that the behaviour remains similar fordifferent equations.

Micron- to nanometer-sized ultrasound agents,like encapsulated microbubbles and echogenic liposomes,are being developed for diagnostic imaging and ultra-sound mediated drug/gene delivery. This review providesan overview of the current state of the art of the mathe-matical models of the acoustic behavior of ultrasound con-trast microbubbles. We also present a review of the in vitroexperimental characterization of the acoustic properties ofmicrobubble based contrast agents undertaken in our lab-oratory. The hierarchical two-pronged approach of model-ing contrast agents we developed is demonstrated for a lipidcoated (SonazoidTM)and a polymer shelled (polyD-L-lactic acid) contrast microbubbles. The acoustic and drugrelease properties of the newly developed echogenic lipo-somes are discussed for their use as simultaneous imagingand drug/gene delivery agents. Although echogenicity is con-clusively demonstrated in experiments, its physical mecha-nisms remain uncertain. Addressing questions raised herewill accelerate further development and eventual clinicalapproval of these novel technologies.

Background: Mechanical stimulation of bone increases bone mass and fracture healing, at least in part, throughincreases in proliferation of osteoblasts and osteoprogenitor cells. Researchers have previously performedin vitrostudies of ultrasound-induced osteoblast proliferation but mostly used fixed ultrasound settings and have reportedwidely varying and inconclusive results. Here we critically investigated the effects of the excitation parameters oflow-intensity pulsed ultrasound (LIPUS) stimulation on proliferation of MC3T3-E1 preosteoblastic cells in monolayercultures.

Methods:We used a custom-designed ultrasound exposure system to vary the key ultrasound parameters—intensity,frequency and excitation duration. MC3T3-E1 cells were seeded in 12-well cell culture plates. Unless otherwise specified,treated cells, in groups of three, were excited twice for 10 min with an interval of 24 h in between after cell seeding.Proliferation rates of these cells were determined using BrdU and MTS assays 24 h after the last LIPUS excitation.All data are presented as the mean ± standard error. The statistical significance was determined using Student'stwo-sample two-tailedttests.

Results:Using discrete LIPUS intensities ranging from 1 to 500 mW/cm2(SATA, spatial average-temporal average), wefound that approximately 75 mW/cm2produced the greatest increase in osteoblast proliferation. Ultrasound exposuresat higher intensity (approximately 465 mW/cm2) significantly reduced proliferation in MC3T3-E1 cells, suggesting thathigh-intensity pulsed ultrasound may increase apoptosis or loss of adhesion in these cells.Variation in LIPUS frequency from 0.5 MHz to 5 MHz indicated that osteoblast proliferation rate was not frequencydependent. We found no difference in the increase in proliferation rate if LIPUS was applied for 30 min/day or 10 min/day, indicating a habituation response.

Conclusion:This study concludes that a short-term stimulation with optimum intensity can enhance proliferation ofpreosteoblast-like bone cells that plays an important role in bone formation and accelerated fracture healing, alsosuggesting a possible therapeutic treatment for reduced bone mass.

Although liposomes are widely used as carriers of drugs and imaging agents, they suffer from a lack ofstability and the slow release of the encapsulated contents at the targeted site. Polymersomes (vesicles ofamphiphilic polymers) are considerably more stable compared to liposomes; however, they alsodemonstrate a slow release for the encapsulated contents, limiting their efficacy as a drug-delivery tool.As a solution, we prepared and characterized echogenic polymersomes, which are programmed torelease the encapsulated drugs rapidly when incubated with cytosolic concentrations of glutathione.These vesicles encapsulated air bubbles inside and efficiently reflected diagnostic-frequency ultrasound.Folate-targeted polymersomes showed an enhanced uptake by breast and pancreatic-cancer cells in amonolayer as well as in three-dimensional spheroid cultures. Polymersomes encapsulated with theanticancer drugs gemcitabine and doxorubicin showed significant cytotoxicity to these cells. Withfurther improvements, these vesicles hold the promise to serve as multifunctional nanocarriers, offeringa triggered release as well as diagnostic ultrasound imaging.

Wall induced lateral migration of a viscoelastic (FENE-MCR) drop in a Newtonianfluid is investigated. Just like a Newtonian drop, a viscoelastic drop reaches a quasi-steady state where the lateral velocity only depends on the instantaneous distancefrom the wall. The drop migration velocity and the deformation scale inversely withthe square and the cube of the distance from the wall, respectively. The migration ve-locity varies non-monotonically with increasing viscoelasticity (increasing Deborahnumber); initially increasing and then decreasing. An analytical explanation has beengiven of the effects by computing the migration velocity as arising from an imagestresslet field due to the drop. The semi-analytical expression matches well with thesimulated migration velocity away from the wall. It contains a viscoelastic stressletcomponent apart from those arising from interfacial tension and viscosity ratio. Themigration dynamics is a result of the competition between the viscous (interfacialtension and viscosity ratio) and the viscoelastic effects. The viscoelastic stressletcontribution towards the migration velocity steadily increases. But the interfacialstresslet—arising purely from the drop shape—first increases and then decreases withrising Deborah number causing the migration velocity to be non-monotonic. The ge-ometric effect of the interfacial stresslet is caused by a corresponding nonmonotonicvariation of the drop inclination. High viscosity ratio is briefly considered to showthat the drop viscoelasticity could stabilize a drop against breakup, and the increase inmigration velocity due to viscoelasticity is larger compared to the viscosity-matchedcase.

Pair-collision between viscous drops in a confined shear is simulated to show that the confinement alters the trajectories of the drops spatially ordering them at a finite separation in the center of the domain. In contrast to free shear where drops eventually adopt free streamlines with a finite cross-stream separation, here they move towards the centerline achieving zero cross-stream separation but a net stream-wise separation. The latter varies as inverse of capillary number and cube of the confinement (distance between the walls). The final stream-wise separation does not depend on the initial positions of the drops when the drops are in the same shear plane. The separation decreases approximately linearly with the initial separation in the vorticity direction. An analytical theory explaining the phenomenon is presented. Effects of the ratio of drop to matrix viscosity are briefly investigated

 

The dynamics of a drop deforming, orienting and moving in a shear flow of aviscoelastic liquid near a wall is numerically investigated using a front-tracking finite-difference method and a semi-analytic theory. The viscoelasticity is modelled usingthe modified FENE-CR constitutive equation. In a Newtonian system, deformation in adrop breaks the reversal symmetry of the system resulting in a migration away fromthe wall. This study shows that the matrix elasticity reduces the migration velocity, thereduction scaling approximately linearly with viscoelasticity (product of the Deborah number De and the ratio of polymer viscosity to total viscosity β). Similar to a Newtonian system, for small Deborah numbers, the dynamics quickly reaches a quasi-steady state where deformation, inclination, as well as migration and slip velocitiesbecome independent of the initial drop–wall separation. They all approximately scaleinversely with the square of the instantaneous separation except for deformation whichscales inversely with the cube of separation. The deformation shows a non-monotonicvariation with increasing viscoelasticity similar to the case of a drop in an unboundedshear and is found to influence little the change in migration. Two competing effectsdue to matrix viscoelasticity on drop migration are identified. The first stems fromthe reduced inclination angle of the drop with increasing viscoelasticity that tries toenhance migration velocity. However, it is overcome by the second effect inhibitingmigration that results from the normal stress differences from the curved streamlinesaround the drop; they are more curved on the side away from the wall comparedwith those in the gap between the wall and the drop, an effect that is also presentfor a rigid particle. A perturbative theory of migration is developed for small ratioof the drop size to its separation from the wall that clearly shows the migrationto be caused by the image stresslet field due to the drop in presence of the wall. The theory delineates the two competing viscoelastic effects, their relative magnitudes, and predicts migration that matches well with the simulation. Using the simulationresults and the stresslet theory, we develop an algebraic expression for the quasi-steadymigration velocity as a function of Ca, De and β. The transient dynamics of themigrating drop is seen to be governed by the finite time needed for development of theviscoelastic stresses. For larger capillary numbers, in both Newtonian and viscoelasticmatrices, a viscous drop fails to reach a quasi-steady state independent of initialdrop–wall separation. Matrix viscoelasticity tends to prevent drop breakup. Drops that† Email address for correspondence: sarkar@gwu.edu

 

 

The stabilizing encapsulation of a microbubble-based ultrasound contrast agent (UCA) critically affectsits acoustic properties. Polymers, which behave differently from materials commonly used (i.e.,lipids or proteins)for monolayer encapsulation, have the potential for better stability and improved control of encapsulation prop-erties. Air-filled microbubbles coated with poly(DL-lactic acid) (PLA) are characterized here usingin vitroacousticexperiments and several models of encapsulation. The interfacial rheological properties of the encapsulation aredetermined according to each model using attenuation of ultrasound through a suspension of microbubbles.Then the model predictions are compared with scattered non-linear (sub- and second harmonic) responses. Forthis microbubble population (average diameter, 1.9mm), the peak in attenuation measurement indicatesa weighted-average resonance frequency of 2.5–3 MHz, which, in contrast to other encapsulated microbubbles,is lower than the resonance frequency of a free bubble of similar size (diameter, 1.9mm). This apparently contra-dictory result stems from the extremely low surface dilational elasticity (around 0.01–0.07 N/m) and the reducedsurface tension of the poly(DL-lactic acid) encapsulation, as well as the polydispersity of the bubble population. Allmodels considered here are shown to behave similarly even in the non-linear regime because of the low surface dila-tional elasticity value. Pressure-dependent scattering measurements at two different excitation frequencies (2.25and 3 MHz) revealed strongly non-linear behavior with 25–30 dB and 5–20 dB enhancements in fundamentaland second-harmonic responses, respectively, for a contrast agent concentration of 1.33mg/mL in the suspension.Sub-harmonic responses are registered above a relatively low generation threshold of 100–150 kPa, with up to 20dB enhancement beyond that pressure. Numerical predictions from all models show good agreement with theexperimentally measured fundamental response, but not with the experimental second-harmonic response. Thecharacteristic features of sub-harmonic responses and the steady response beyond the threshold are matchedwell by model predictions. However, prediction of the threshold value depends on estimated properties and sizedistribution. The variation in size distribution from sample to sample leads to variation in estimates of encapsula-tion properties: the lowest estimated value for surface dilational viscosity better predicts the sub-harmonicthreshold.

Although lipid nanoparticles are promising drugdelivery vehicles, passive release of encapsulated contents atthe target site is often slow. Herein, we report contents releasefrom targeted, polymer-coated, echogenic lipid nanoparticles inthe cell cytoplasm by redox trigger and simultaneouslyenhanced by diagnostic frequency ultrasound. The lipidnanoparticles were polymerized on the external leaflet usinga disulfide cross-linker. In the presence of cytosolicconcentrations of glutathione, the lipid nanoparticles released76% of encapsulated contents. Plasma concentrations ofglutathione failed to releasethe encapsulated contents.Application of 3 MHz ultrasound for 2 min simultaneouslywith the reducing agent enhanced the release to 96%. Folicacid conjugated, doxorubicin-loaded nanoparticles showed enhanced uptake and higher cytotoxicity in cancer cells overexpressingthe folate receptor (compared to the control). With further developments, these lipid nanoparticles have the potential to be usedas multimodal nanocarriers for simultaneous targeted drug delivery and ultrasound imaging.

7 in Year 2011-2012

A recent study [Katiyar and Sarkar (2011). J. Acoust. Soc. Am.130, 3137–3147] showed that incontrast to the analytical result for free bubbles, the minimum threshold for subharmonic generationfor contrast microbubbles does not necessarily occur at twice the resonance frequency. Hereincreased damping—either due to the small radius or the encapsulation—is shown to shift the mini-mum threshold away from twice the resonance frequency. Free bubbles as well as four modelsof the contrast agent encapsulation are investigated varying the surface dilatational viscosity.Encapsulation properties are determined using measured attenuation data for a commercial contrastagent. For sufficiently small damping, models predict two minima for the threshold curve—one attwice the resonance frequency being lower than the other at resonance frequency—in accord withthe classical analytical result. However, increased damping damps the bubble response more attwice the resonance than at resonance, leading to a flattening of the threshold curve and a gradualshift of the absolute minimum from twice the resonance frequency toward the resonance frequency.The deviation from the classical result stems from the fact that the perturbation analysis employedto obtain it assumes small damping, not always applicable for contrast microbubbles.

Echogenic liposomes (ELIP) are an excellent candidate for concurrent imaging and drug delivery applica-tions. They combine the advantages of liposomes-biocompatibility and ability to encapsulate both hydro-phobic and hydrophilic drugs-with strong reflections of ultrasound. The objective of this study is toperform a detailedin vitroacoustic characterization – including nonlinear scattering that has not beenstudied before – along with an investigation of the primary mechanism of echogenicity. Both componentsare critical for developing viable clinical applications of ELIP. Mannitol, a cryoprotectant, added duringthe preparation of ELIP is commonly believed to be critical in making them echogenic. Accordingly, hereELIP prepared with varying amount of mannitol concentration are investigated for their pressure depen-dent linear and non-linear scattered responses. The average diameter of these liposomes is measured tobe 125–185 nm. But they have a broad size distribution including liposomes with diameters over a micro-meter as observed by TEM and AFM. These larger liposomes are critical for the overall echogenicity.Attenuation through liposomal solution is measured with four different transducers (central frequencies2.25, 3.5, 5, 10 MHz). Measured attenuation increases linearly with liposome concentration indicatingabsence of acoustic interactions between liposomes. Due to the broad size distribution, the attenuationshows a flat response without a distinct peak in the range of frequencies (1–12 MHz) investigated. A15–20 dB enhancement with 1.67lg/ml of lipids is observed both for the scattered fundamental andthe second harmonic responses at 3.5 MHz excitation frequency and 50–800 kPa amplitude. It demon-strates the efficacy of ELIP for fundamental as well as harmonic ultrasound imaging. The scatteredresponse however does not show any distinct subharmonic peak for the acoustic excitation parametersstudied. Small amount of mannitol proves critical for echogenicity. However, mannitol concentrationabove 100 mM shows no effect.

The extracellular enzyme matrix metalloproteinase-9(MMP-9) is overexpressed in atherosclerotic plaques and in metastaticcancers. The enzyme is responsible for rupture of the plaques and forthe invasion and metastasis of a large number of cancers. The ability ofultrasonic excitation to induce thermal and mechanical effects has beenused to release drugs from different carriers. However, the majority ofthese studies were performed with low frequency ultrasound (LFUS) atkilohertz frequencies. Clinical usage of LFUS excitations will be limiteddue to harmful biological effects. Herein, we report our results on therelease of encapsulated contents from substrate lipopeptide incorpo-rated echogenic liposomes triggered by recombinant human MMP-9.The contents release was further enhanced by the application ofdiagnostic frequency (3 MHz) ultrasound. The echogenic liposomeswere successfully imaged employing a medical ultrasound transducer(4−15 MHz). The conditioned cell culture media from cancer cells (secreting MMP-9) released the encapsulated dye from theliposomes (30−50%), and this release is also increased (50−80%) by applying diagnostic frequency ultrasound (3 MHz) for 3min. With further developments, these liposomes have the potential to serve as multimodal carriers for triggered release andsimultaneous ultrasound imaging.

Deformation and sedimentation velocities of a viscoelastic drop falling through a Newtonianmedium are numerically investigated using a front-tracking finite difference method. In contrast toa viscous drop, viscoelasticity deforms an initially spherical drop into an oblate shape and decreasesits sedimentation velocity. Further increase of elasticity results in a dimple at the rear end, as theviscoelastic stress at the trailing end of the drop pulls the drop interface inward. The dimplebecomes more prominent with increasing Deborah number, amount of polymeric viscosity, andcapillary number. An approximate analysis is performed to model the stress development along theaxis of symmetry, specifically its increase at the rear end that governs the dimple formation. Foreven higher values of Deborah number, the interfacial tension cannot balance the viscoelasticstresses leading to an unstable situation toward a toroidal shape. We numerically find the criticalDeborah number for the transition. It shows an approximate inverse scaling with capillary number.For unstable cases, downward progressing dimple develops a globular end. Development of theglobular end results in a sudden increase in the cross-sectional area of the drop and a sharp decreaseof the settling velocity.

Variation of subharmonic response from contrast microbubbles with ambient pressure is numericallyinvestigated for non-invasive monitoring of organ-level blood pressure. Previously, several contrastmicrobubbles bothin vitroandin vivoregistered approximately linear (5–15 dB) subharmonicresponse reduction with 188 mm Hg change in ambient pressure. In contrast, simulated subharmonicresponse from a single microbubble is seen here to either increase or decrease with ambient pressure.This is shown using the code BUBBLESIM for encapsulated microbubbles, and then the underlyingdynamics is investigated using a free bubble model. The ratio of the excitation frequency to the natu-ral frequency of the bubble is the determining parameter—increasing ambient pressure increases nat-ural frequency thereby changing this ratio. For frequency ratio below a lower critical value,increasing ambient pressure monotonically decreases subharmonic response. Above an upper criticalvalue of the same ratio, increasing ambient pressure increases subharmonic response; in between, thesubharmonic variation is non-monotonic. The precise values of frequency ratio for these three differ-ent trends depend on bubble radius and excitation amplitude. The modeled increase or decrease ofsubharmonic with ambient pressure, when one happens, is approximately linear only for certain rangeof excitation levels. Possible reasons for discrepancies between model and previous experiments arediscussed.

Deformation of a viscous drop in shear at finite inertia and the streamlines around itare numerically investigated. Inertia destroys the closed streamlines found in Stokesflow. It creates reversed streamlines and streamlines spiralling around the vorticityaxis. Spiralling streamlines spiral either towards the central shear plane or away fromit depending on the viscosity ratio and the inertia. The zones of open or reversedstreamlines as well as streamlines spiralling towards or away from the central shearplane are delineated for varying viscosity ratio and Reynolds number. In contrast tothe infinite extent of the closed Stokes streamlines around a rigid sphere in shear, theregion of the spiralling streamlines in the vorticity direction both for a rigid sphere anda drop shrinks with inertia. Inertia increases deformation, and introduces oscillations indrop shape. An approximate analysis explains the scaling of oscillation frequency anddamping with Reynolds and capillary numbers. The steady-state drop inclination anglewith the flow axis increases with increasing Reynolds number for small Reynoldsnumber. But it decreases at higher Reynolds number, especially for larger capillarynumbers. For smaller capillary numbers, drop inclination reaches higher than 45?(thedirection of maximum extension), critically affecting the interfacial stresses due tothe drop. It changes the sign of first and second normal interfacial stress differences(and thereby these components of the effective stresses of an emulsion of such drops).Increasing viscosity ratio orients the drop towards the flow axis, which increases thecritical Reynolds number above which the drop inclination reaches more than 45?.

Six models of contrast microbubbles are investigated to determine the excitation threshold for sub-harmonic generation. The models are applied to a commercial contrast agent; its characteristic pa-rameters according to each model are determined using experimentally measured ultrasoundattenuation. In contrast to the classical perturbative result, the minimum threshold for subharmonicgeneration is not always predicted at excitation with twice the resonance frequency; instead itoccurs over a range of frequencies from resonance to twice the resonance frequency. The quantita-tive variation of the threshold with frequency depends on the model and the bubble radius. All mod-els are transformed into a common interfacial rheological form, where the encapsulation isrepresented by two radius dependent surface properties—effective surface tension and surface dila-tational viscosity. Variation of the effective surface tension with radius, specifically having anupper limit (resulting from strain softening or rupture of the encapsulation during expansion), playsa critical role. Without the upper limit, the predicted threshold is extremely large, especially nearthe resonance frequency. Having a lower limit on surface tension (e.g., zero surface tension in thebuckled state) increases the threshold value at twice the resonance frequency, in some cases shiftingthe minimum threshold toward resonance.

11 in Year 2008 -2010

Effects of drop and matrix viscoelasticity on the retraction of a sheared drop are numerically investigated.Retraction of an Oldroyd-B drop in a Newtonian matrix is initially faster and later slower with increasingdrop Deborah number. The observed behavior is explained using an ordinary differential equation modelrepresenting the dominant balance between various forces during retraction. The initial faster relaxationof viscoelastic drops is due to viscoelastic stresses pulling the drop interface at the tips inward. The laterslower retraction is due to the slowly-relaxing viscoelastic forces at the equator, where they act againstthe capillary force. The drop inclination decreases substantially during retraction unlike in a Newtoniancase. Matrix viscoelasticity slows the relaxation of a Newtonian drop because of the increasingly slowrelaxation of highly stretched polymers near the drop tip with increasing Deborah number. Increasingthe ratio of polymeric to total viscosity further accentuates the viscoelastic effects in both cases. For anOldroyd-B drop in an Oldroyd-B matrix, a competition between the dispersed and the continuous phaseelasticities, represented by their ratio, determines the dynamics; larger values of the ratio leads again toinitial faster and later slower retraction.

Linear stability analysis is performed for a mathematical model of diffusion of gases from an encapsulatedmicrobubble. It is an Epstein–Plesset model modified to account for encapsulation elasticity and finite gaspermeability. Although bubbles, containing gases other than air, are considered, the final stable bubble, ifany, contains only air, and stability is achieved only when the surrounding medium is saturated or over-saturated with air. In absence of encapsulation elasticity, only a neutral stability is achieved for zero sur-face tension, the other solution being unstable. For an elastic encapsulation, different equilibriumsolutions are obtained depending on the saturation level and whether the surface tension is smaller orhigher than the elasticity. For an elastic encapsulation, elasticity can stabilize the bubble. However,imposing a non-negativity condition on the effective surface tension (consisting of reference surface ten-sion and the elastic stress) leads to an equilibrium radius which is only neutrally stable. If the encapsu-lation can support a net compressive stress, it achieves actual stability. The linear stability results areconsistent with our recent numerical findings. Physical mechanisms for the stability or instability of var-ious equilibriums are provided.

Two nonlinear interfacial elasticity models—interfacial elasticity decreasing linearly andexponentially with area fraction—are developed for the encapsulation of contrast microbubbles. Thestrain softeningdecreasing elasticityresults from the decreasing association between theconstitutive molecules of the encapsulation. The models are used to find the characteristic propertiessurface tension, interfacial elasticity, interfacial viscosity and nonlinear elasticity parametersfor acommercial contrast agent. Properties are found using the ultrasound attenuation measured througha suspension of contrast agent. Dynamics of the resulting models are simulated, compared withother existing models and discussed. Imposing non-negativity on the effective surface tensiontheencapsulation experiences no net compressive stressshows “compression-only” behavior. Theexponential and the quadraticlinearly varying elasticitymodels result in similar behaviors. Thevalidity of the models is investigated by comparing their predictions of the scattered nonlinearresponse for the contrast agent at higher excitations against experimental measurement. All modelspredict well the scattered fundamental response. The nonlinear strain softening included in theproposed elastic models of the encapsulation improves their ability to predict subharmonic response.They predict the threshold excitation for the initiation of subharmonic response and its subsequentsaturation.

Interactions between a pair of equal-size viscous drops in shear are numerically investigated at finiteReynolds numberRe=0.1–10. At low Reynolds number the simulation compares well with aprevious experimental observation. Apart from the usual pairwise motion where drops driven byshear pass over each othertype I trajectory, finite inertia introduces a new typetype IIofreversed trajectory where drops approaching each other reverse their initial trajectories. The newtrajectory is explained by a reversed streamline pattern observed around a single drop in an imposedshear, and is similar to what is also observed for rigid spheres at finite inertia. However, dropdeformability introduces a nonuniform transition from one to the other type of trajectory—dropsdisplay type I trajectory for high and low capillary numbers and type II for intermediate capillarynumbers. The phenomenon is explained by noting that increasing capillary number gives rise tocompeting effects—while it increases drop deformation and therefore increases resistance to slidingmotion, it also increases drop flexibility, decreases inclination angle, and overall effect of the drop’spresence is reduced, all helping them to slide by. The nonuniform behavior—type II trajectory foran intermediate range of capillary numbers—occurs only at Reynolds number above a critical value.Further increase in Reynolds number increases the range of capillary numbers for type II trajectory.For type I trajectory, terminal cross-stream separation increases linearly with increasing inertiaindicating an enhanced shear induced diffusion. Increasing initial streamwise separation aids inreversedtype IItrajectory due to increased overlap with the reversed streamline zone. Increasingcross-stream distance expectedly facilitatestype Isliding motion. For passing dropstype Itrajectory, terminal cross-stream separation is not appreciably affected by capillary number andinitial drop separation.

Deformation of an Oldroyd B drop in a Newtonian matrix under steady shear is simulated using a fronttracking finite difference method for varying viscosity ratio. For drop viscosity lower than that of thematrix, the long-time steady deformation behavior is similar to that of the viscosity matched system—thedrop shows reduced deformation with increasing Deborah number due to the increased inhibiting vis-coelastic normal stress inside the drop. However for higher viscosity ratio systems, the drop response isnon-monotonic—the steady drop deformation first decreases with increasing Deborah number but abovea critical Deborah number, it increases with further increase in Deborah number, reaching higher thanthe viscous case value for some viscosity ratios. We explain the increase in deformation with Deborahnumber by noting that at higher viscosity ratios, strain rate inside the drop is reduced, thereby reducingthe inhibiting viscoelastic stress. Furthermore, similar to the viscosity matched system, the drop inclina-tion angle increases with increasing Deborah number. A drop aligned more with the maximum stretchingaxis at 45 degree of the imposed shear, experiences increased viscous stretching. With increased ratioof polymeric viscosity to total drop viscosity, the drop deformation decreases and the inclination angleincreases. Our simulation results compare favorably with a number of experimental and computationalresults from other researchers.

Gas diffusion from an encapsulated microbubble is modeled using an explicit linear relation for gaspermeation through the encapsulation. Both the cases of single gas (air) and multiple gases (perfluorocarbon insidethe bubble and air dissolved in surrounding liquid) are considered. An analytical expression for the dissolutiontime for an encapsulated air bubble is obtained; it showed that for small permeability the dissolution time increaseslinearly with decreasing permeability. A perfluorocarbon-filled contrast microbubble such as DefinityÒwas pre-dicted to experience a transient growth because of air infusion before it dissolves in conformity with previousexperimental findings. The growth phase occurs only for bubbles with a critical value of initial mole fraction ofperfluorocarbon relative to air. With empirically obtained property values, the dissolution time of a 2.5-microndiameter (same as that of Definity), lipid-coated octafluoropropane bubble, with surface tension 25 mN/m, is pre-dicted to be 42 min in an air-saturated medium. The properties such as shell permeability, surface tension andrelative mole fraction of octafluoropropane are varied to investigate their effects on the time scales of bubblegrowth and dissolution, including their asymptotic scalings where appropriate. The dissolution dynamics scaleswith permeability, in that when the time is nondimensioanlized with permeability, curves for different permeabil-ities collapse on a single curve. Investigation of bubbles filled with other gases (nonoctafluoropropane perfluoro-carbon and sulfur hexafluoride) indicates longer dissolution time because of lower solubility and lowerdiffusivity for larger gas molecules. For such micron-size encapsulated bubbles, lifetime of hours is possibleonly at extremely low surface tension (,1 mN/m) or at extreme oversaturation.(E-mail:sarkar@udel.edu)Ó2009 World Federation for Ultrasound in Medicine & Biology.

Drops driven toward each other by shear at finite inertia follow two distinct types of trajectories.Type I trajectory is similar to the one in Stokes flow where drops slide past each other. However, atfinite inertia, drops display a new type II trajectory, where they reverse their paths. Increasingviscosity ratio results in a transition from type II to type I trajectory. The transition is caused bydecreased drop deformation and increased alignment with the flow at higher drop viscosity; bothdecrease the zone of reversed streamlines that accompanies a drop at finite inertia. The transition isdelineated in a phase diagram of Reynolds number and viscosity ratio for different capillarynumbers. The critical viscosity ratio, where a type II transitions into type I, increases with Reynoldsnumber except at higher capillary numbers, where the critical viscosity ratio shows a slightnonmonotonic variation with Reynolds number. Also, it is nonmonotonic with capillary numbers inthat for a fixed Reynolds number, the critical viscosity ratio first increases with increasing capillarynumber and then decreases. Similar to the Stokes regime, increased viscosity ratio leads to adecreased postcollision cross-stream separation effectively decreasing the shear induced diffusion.Higher viscosity ratio results in an increased separation between drops during encounter, whichresults in a smaller interaction time. With drops placed initially at different shear planes, drops comeunder the influence of the reversed flow zone around a single drop that broadens off the central shearplane. Consequently, the trajectory changes from type I to type II as the offset in the vorticitydirection increases. The change depends on the initial offset in the shear direction as well. The finaldisplacement in the shear direction varies linearly with the initial offset. The net relativedisplacement in the shear direction shows a gradual decrease with increasing offset. The net relativedisplacement in the vorticity direction with increasing offset first increases from a zero value whendrops are placed at the same shear plane to a maximum and then decreases. For certain cases, itreaches a negative value.

A model for gas transport from an encapsulated microbubble into the surrounding medium is developedand investigated incorporating the effects of encapsulation elasticity. Encapsulation elasticity stabilizesmicrobubbles against dissolution and explains the long shelf life of microbubble contrast agent. We con-sider air bubbles as well as bubbles containing perfluorocarbon gas. Analytical conditions between satu-ration level, surface tension and interfacial dilatational elasticity are determined for attaining non-zeroequilibrium radius for these microbubbles. Numerical solution of the equation verifies the stability ofthe equilibrium radii. In an undersaturated medium all encapsulated bubbles dissolve. In a saturatedmedium, an encapsulated bubble is found to achieve a long-time stable radius when interfacial dilata-tional elasticity is larger than equilibrium surface tension. For bubbles with interfacial dilatational elas-ticity smaller than the equilibrium surface tension, stable bubble of non-zero radius can be achieved onlywhen the saturation level is greater than a critical value. Even if they initially contain a gas other than air,bubbles that reach a stable radius finally become air bubbles. The model is applied to an octafluoropro-pane filled lipid-coated 2.5lm bubble, which displayed a transient swelling due to air intake beforereaching an equilibrium size. Effects of elasticity, shell permeability, initial mole fraction, initial radiusand saturation level are investigated and discussed. Shell permeability and mole fraction do not affectthe final equilibrium radius of the microbubble but affect the time scale and the transient dynamics. Sim-ilarly, the ratio of equilibrium radius to initial radius remains unaffected by the variation in initial radius.

The deformation of a Newtonian/viscoelastic drop suspended in a viscoelastic fluidis investigated using a three-dimensional front-tracking finite-difference method.The viscoelasticity is modelled using the Oldroyd-B constitutive equation. Matrixviscoelasticity affects the drop deformation and the inclination angle with the flowdirection. Numerical predictions of these quantities are compared with previousexperimental measurements using Boger fluids. The elastic and viscous stresses atthe interface, polymer orientation, and the elastic and viscous forces in the domainare carefully investigated as they affect the drop response. Significant change in thedrop inclination with increasing viscoelasticity is observed; this is explained in termsof the first normal stress difference. A non-monotonic change – a decrease followedby an increase – in the steady-state drop deformation is observed with increasingDeborah number (De) and explained in terms of the competition between increasedlocalized polymer stretching at the drop tips and the decreased viscous stretchingdue to change in drop orientation angle. The transient drop orientation angle isfound to evolve on the polymer relaxation time scale for highDe. The breakup ofa viscous drop in a viscoelastic matrix is inhibited for smallDe, and promoted athigherDe. Polymeric to total viscosity ratioβwas seen to affect the result throughthe combined parameterβDeindicating a dominant role of the first normal stressdifference. A viscoelastic drop in a viscoelastic matrix with matched relaxation timeexperiences less deformation compared to the case when one of the phases is viscous;but the inclination angle assumes an intermediate value between two extreme cases.Increased drop phase viscoelasticity compared to matrix phase leads to decreaseddeformation.

The dynamics of a liquid capsule enclosed by an elastic membrane in a shear flow is investigated using a front trackingfinite difference method. We compute deformation, orientation and tank-treading of the capsule, as functions of the forcing(capillary number) and the viscosity ratio for two different membrane constitutive equations – Neo-Hookean and Skalak.The computed results compare very well with those obtained by high-order boundary element methods as well as the smalldeformation perturbation analysis. The simulation shows that a drop and a capsule, even under those circumstances thatresult in the same Taylor deformation criterion for both, attain very different shapes. The tank-treading period even fordifferent capillary numbers as well as capsules with different constitutive laws, is primarily determined by the deformationand the viscosity ratio. At low capillary numbers the simulation predicts buckling due to large compressive stresses on themembrane. However, we show that in shear, unlike in extension, the tank-treading motion can inhibit the buckling insta-bility and gives rise to a stable evolution even in presence of membrane compressive stresses. At large capillary numbers thecapsule experiences large bounded shape followed by tip buckling indicating possible membrane breakup.Ó2008 Elsevier Inc. All rights reserved.

Steady shear rheology of a dilute emulsion with viscoelastic inclusions is numerically investigated using direct numerical simulations. Batchelor’sformulation for rheology of a viscous emulsion is extended for a viscoelastic system. Viscoelasticity is modeled using the Oldroyd-B constitutiveequation. A front-tracking finite difference code is used to numerically determine the drop shape, and solve for the velocity and stress fields. Theeffective stress of the viscoelastic emulsion has three different components due to interfacial tension, viscosity difference (not considered here) andthe drop phase viscoelasticity. The interfacial contributions – first and second normal stress differences and shear stresses – vary with Capillarynumber in a manner similar to those of a Newtonian system. However the shear viscosity decreases with viscoelasticity at low Capillary numbers,and increases at high Capillary numbers. The first normal stress difference due to interfacial contribution decreases with increasing drop phaseviscoelasticity. The first normal stress difference due to the drop phase viscoelasticity is found to have a complex dependence on Capillary andDeborah numbers, in contrast with the linear mixing rule. Drop phase viscoelasticity does not contribute significantly to effective shear viscosityof the emulsion. The total first normal stress difference shows an increase with drop phase viscoelasticity at high Capillary numbers. However atlow Capillary numbers, a non-monotonic behavior is observed. The results are explained by examining the stress field and the drop shape.© 2007 Elsevier B.V. All rights reserved.

10 in Year 2005 -Year 2007

The deformation of a viscoelastic drop suspended in a Newtonian fluid subjectedto a steady shear is investigated using a front-tracking finite-difference method.The viscoelasticity is modelled using the Oldroyd-B constitutive equation. The dropresponse with increasing relaxation timeλand varying polymeric to the total dropviscosity ratioβis studied and explained by examining the elastic and viscousstresses at the interface. Steady-state drop deformation was seen to decrease from itsNewtonian value with increasing viscoelasticity. A slight non-monotonicity in steady-state deformation with increasing Deborah number is observed at high Capillarynumbers. Transient drop deformation displays an overshoot before settling downto a lower value of deformation. The overshoot increases with increasingβ.Thedrop shows slightly decreased alignment with the flow with increasing viscoelasticity.A simple ordinary differential equation model is developed to explain the variousbehaviours and the scalings observed numerically. The critical Capillary number fordrop breakup is observed to increase with Deborah number owing to the inhibitiveeffects of viscoelasticity, the increase being linear for small Deborah number.

Two-dimensional simulations of fl ow instability at the interface of a three-layer, density-matched, viscosity-stratifi ed Poiseuille fl ow are performed using a front-tracking/fi nite difference method. This is an extension of the study for the stability of two-layer viscosity-stratifi ed fl ow of Cao et al., Int. J. Multiphase Flow, 30, 1485-1508 (2004). We present results for large-amplitude non-linear evolution of the interface for varying viscos-ity ratio m, Weber number We, and phase difference between the perturbations of the two interfaces. Strong non-linear behaviour is observed for relatively large initial perturbation amplitude. The higher viscosity fl uid is drawn out as a fi nger that penetrates into the lower viscosity layer. The fi nger originates at the crest of the perturbation at the interface. The simulated interface shape compares well with previously reported experiments. Increasing interfacial tension retards the growth rate of the interface as expected, whereas increasing the viscosity ratio enhances it. The sinuous instability appears to evolve faster than the varicose one. For certain fl ow parameters the high-viscosity fi nger displays a bulbous tip, which is also seen in our previously conducted experiments and two-layer results, although it is less pronounced. The low-viscosity intruding fi nger does not display this curious bulbous tip. Drop formation is precluded by the two-dimensional nature of the calculations

Deformation and breakup of a viscous drop in a potential vortex are numericallysimulated. Capillary number, Reynolds number, and viscosity and density ratios arevaried to investigate their effects on the drop dynamics. The vortex locally gives riseto an extensional flow near the drop with the axis of extension rotating at a constantrate, as the drop revolves around the vortex centre. The rotation of the axis plays acritical role in the competing dynamics between the flow-induced stretching and theinterfacial tension. The relation between the rotating extensional flow and a shearflow is explored. For low capillary numbers, a periodic state is reached, where thedrop deforms into an ellipsoidal shape and undergoes steady rotation with a distinctphase lag behind the imposed flow. For density-and-viscosity-matched drops, increasedinterfacial tension results in decreased deformation and reduced phase lag. Increasedinertia promotes deformation. In the presence of inertia, decreasing capillary numberleads to a negative phase lag. The rotation of the extension axis inhibits deformation at low values of the Reynolds number. But at high Reynolds numbers, rotation-induced centrifugal forces promote deformation. At low and high viscosity ratios, an increasein viscosity ratio leads to enhancement and reduction in deformation, respectively. At density ratios larger than unity, the drop deformation displays resonance inthat it varies non-monotonically with a distinct peak with variation of interfacialtension and density ratio. The peak corresponds to the natural frequency of thedrop deformation matching with the frequency of rotation due to the vortex. Asimple physical model is used to explain various observations including asymptoticscalings. We also explore different mechanisms for drop breakup at different Reynoldsnumber, and provide critical capillary numbers as functions of other parameters.In particular, vortex-induced resonance offers an alternative mechanism for size-selective drop breakup. Details of flow fields and transients are also presented and discussed.

Zero-thickness interface models are developed to describe the encapsulation of microbubblecontrast agents. Two different rheological models of the interface, Newtonianviscousandviscoelastic, with rheological parameters such as surface tension, surface dilatational viscosity, andsurface dilatational elasticity are presented to characterize the encapsulation. The models are appliedto characterize a widely used microbubble based ultrasound contrast agent. Attenuation ofultrasound passing through a solution of contrast agent is measured. The model parameters for thecontrast agent are determined by matching the linearized model dynamics with measuredattenuation data. The models are investigated for its ability to match with other experiments.Specifically, model predictions are compared with scattered fundamental and subharmonicresponses. Experiments and model prediction results are discussed along with thoseobtained using an existing modelChurch, J. Acoust. Soc. Am.97, 15101995and Hoffet al.,J. Acoust. Soc. Am.107, 22722000of contrast agents.

The rheology of a dilute emulsion of viscous drops in an oscillating extensional flow is investigated. Deforming drop shape is computedusing a front tracking finite difference method. Excess stresses due to drops are determined using Bachelor’s formula neglecting drop–dropinteractions. We present and discuss the relations between the excess stress and the applied strain rate. We explore the linear extensionalrheology by computing extensional storage and loss moduli. The effects of frequency and surface tension variations are discussed andcompared with analytical models of Oldroyd and Yu and Bousmina. We find that the nature of the excess interfacial stress depends on therelative magnitudes of the time period of oscillation and the relaxation time of the droplet. The excess stress is predominantly elastic (viscous)if the period is much smaller (larger) than the relaxation time. These phenomena are explained using the detail drop dynamics.

Broadband attenuation measurement has been widely used for characterizing ultrasound contrastagents. Chen et al. (2002) recently suggested that broadband attenuation data depend on the center frequency ofthe broadband excitation pulse and, therefore, that they are not a reliable measure of the bubble behavior. Weinvestigated the suitability of measurement of broadband attenuation as a characterizing tool using the contrastagent Definity®as a test case. Analyzing the attenuation data obtained with three broadband unfocusedtransducers with different center frequencies (2.25, 3.5 and 5 MHz), we found that attenuation is independent ofthe transducer used and matches in the overlap regions of any two transducers. Attenuation does not depend onexcitation pressure amplitude as long as the excitation amplitude remains below a critical value (0.26 MPa),indicating that the measurement of broadband attenuation below critical excitation can, indeed, be used forcharacterization. Furthermore, the linear relationship of attenuation with concentrations of Definity®is alsoinvestigated.

A viscous drop deforming in a planar oscillating extensional flow is numerically simulated using afront-tracking finite-difference method. The effects of periodic forcing and interfacial tension arestudied at low but finite inertia. The oscillation leads to decreased deformation and bounded dropshapes for conditions for which steady extension results in drop breakup. The drop displays aresonance phenomenon where the deformation reaches a maximum when the forcing frequencymatches the natural frequency of the drop. The large deformation at resonance indicates a possiblemechanism for size selective breakup by flows with appropriate fluctuation frequency. The detailstructure of the flow at different time instants within a period for various values of interfacial tensionand frequency is investigated. The drop dynamics shows a complex phase relation with the forcingflow. Competition between the inertia-induced dynamic pressure and the viscous stresses leads toboth positive and negative values of the phase and a complex variation with interfacial tension andforcing frequency. A second-order ordinary differential equation model with appropriaterepresentation of the pressure and viscous forces is developed that qualitatively explains the phasebehaviors. For the highest inertia case considered in this papersRe = 10.0d, the drop dynamicsbecomes aperiodic at resonance marked by a strong subharmonic component in the frequencyspectrum.

The relation between the normal stress and the imposed strain for a Newtonian emulsion in anoscillating extensional flow is computed at finite Reynolds numbers using numerically simulated dropgeometry. The interfacial stress was determined using Batchelor’s formalism. In the presence of inertia, the phase between the stress and the strain deviates from Stokes’s flow, and leads to a negative elastic modulus at small frequencies. The results are explained by a mass-spring-dashpot model.

Effects of inertia on the rheology of dilute Newtonian emulsion of drops in shear flow areinvestigated using direct numerical simulation. The drop shape and flow are computed by solvingthe Navier-Stokes equation in two phases using Front-tracking method. Effective stress iscomputed using Batchelor’s formulation, where the interfacial stress is obtained from the simulateddrop shape and the perturbation stress from the velocity field. At low Reynolds number, thesimulation shows good agreement with various analytical results and experimental measurements.At higher inertia deformation is enhanced and the tilt angle of the drop becomes larger thanforty-five degree. The inertial morphology directly affects interfacial stresses. The first and thesecond interfacial normal stress differences are found to change sign due to the change in droporientation. The interfacial shear stress is enhanced by inertia and decreases with capillary numberat lower inertia but increases at higher inertia. The total excess stresses including perturbationstress contribution shows similar patterns

Micron-size bubbles encapsulated by a stabilizing layer of surface-active materials are used inmedical ultrasound imaging and drug delivery. Their destruction stimulated by ultrasoundin vivoplays a critical role in both applications. We investigate the destruction process of microbubbles ina commercially available contrast agent by measuring the attenuation of ultrasound through it. Themeasurement is performed with single-cycle bursts from an unfocused transducerwith a centerfrequency of 5 MHzfor varying pressure amplitudes at 50-, 100-, and 200-Hz pulse repetitionfrequenciesPRFwith duty cycles 0.001%, 0.002%, and 0.004%, respectively. At low excitation,the attenuation is found to increase with time. With increased excitation level, the attenuation leveldecreases with time, indicating destruction of microbubbles. There is a critical pressure amplitude1.2 MPafor all three PRFs, below which there is no significant bubble destruction. Above thecritical pressure amplitudes the rate of destruction depends on excitation levels. But at high-pressureamplitudes the destruction becomes independent of excitation pressure amplitude. The results areinterpreted to identify two different mechanisms of bubble destruction by its signature inattenuation, namely, slow dissolution by diffusion and catastrophic shell rupture. The differentmodes are discussed in detail with their implications in medical applications

4 in Year 2001-Year 2004

Two-dimensional simulations of flow instability at the interface of a two-layer, density-matched, viscos-ity-stratified Poiseuille flow are performed using a front-tracking/finite difference method. We presentresults for the small-amplitude (linear) growth rate of the instability at small to medium Reynolds numberfor varying thickness ration, viscosity ratiom, and wavenumber. We also present results for large-ampli-tude non-linear evolution of the interface for varying viscosity ratio and interfacial tension. For the linearcase, the interfacial mode is neutrally stable forn¼ffiffiffiffimpas predicted by analysis. The growth rate is pro-portional to Reynolds number for smallRe, and increases with viscosity ratio. The growth rate alsoincreases when the thickness of the more viscous layer is reduced. Strong non-linear behavior is observedfor relatively large initial perturbation amplitude. The higher viscosity fluid is drawn out as a finger thatpenetrates into the lower viscosity layer. The simulated interface shape compares well with previouslyreported experiments. Increasing interfacial tension retards the growth rate of the interface as expected,whereas increasing the viscosity ratio enhances it. Drop formation at the small Reynolds number consid-ered in this study is precluded by the two-dimensional nature of the calculations.

A quantitative model of the dynamics of an encapsulated microbubble contrast agent will be avaluable tool in contrast ultrasound (US). Such a model must have predictive ability for widely varyingfrequencies and pressure amplitudes. We have developed a new model for contrast agents, and successfullyinvestigated its applicability for a wide range of operating parameters. The encapsulation is modeled as acomplex interface of an infinitesimal thickness. A Newtonian rheology with surface viscosities and interfacialtension is assumed for the interface, and a modified Rayleigh–Plesset equation is derived. The rheologicalparameters (surface tension and surface dilatational viscosity) for a number of contrast agents (Albunex,Optisonand Quantison) are determined by matching the linearized model dynamics with experimentallyobtained attenuation data. The model behavior for Optison(surface tension 0.9 N/m and surface dilatationalviscosity 0.08 msP) was investigated in detail. Specifically, we have carried out a detailed interrogation of themodel, fitted in the linear regime, for its nonlinear prediction. In contrast to existing models, the new model isfound to capture the characteristic subharmonic emission of Optisonobserved by Shi et al. (1999). A detailedparametric study of the bubble behavior was executed using the ratio of scattering to attenuation (STAR). Itshows that the encapsulation drastically reduces the influence of resonance frequency on scattering cross-section,suggesting possible means of improvement in imaging at off-resonant frequencies. The predictive capability of thepresent model indicates that it can be used for characterizing different agents and designing new ones.

An inertialess jet containing Newtonian drops in a gravitation-free field has been modeled by incorporating thedetailed micro-structural dynamics. Interactions of the drops with the continuous phase, with the wall, and with thejet surface are accounted for, thereby eliminating the need for additional constitutive assumptions. The deformationof the jet and drops, and the dispersion of drops are predicted. The present paper serves two purposes. First, acomplete description of the modeling process and its limitation is presented. Second, preliminary results for singleand for small clusters of drops indicate the potential inherent in this computational approach. The model flow issolved in two dimensions using the boundary element method (BEM). Effects of different drop parameters, such astheir number, relative position, and interfacial tension are investigated.

In Sarkar & Schowalter (2001), we reported results from numerical simulations of dropdeformation in various classes of time-periodic straining flows at non-zero Reynoldsnumber. As often occurs, analytical solutions provide more e ective understanding ofthe structure and signi cance of a phenomenon. Here we describe drop deformationpredicted from analytical solutions to linear time-periodic straining flows. Threedi erent limiting cases are considered: an unsteady Stokes flow that retains all butthe nonlinear advection terms, a Stokes flow that neglects inertia altogether, and aninviscid potential flow. The rst limit is in clear contrast to the common approachin emulsion literature that resorts almost always to the Stokes flow assumption.The analysis clearly shows the forced{damped mass{spring system underlying thephysical phenomena, which distinguishes it from the inertialess Stokes flow. Thepotential flow also depicts resonance, albeit of an undamped system, and provides animportant limit of the problem. The drop deformation is assumed to be small, and aperturbative approach has been employed. The rst-order problem has been solvedto arrive at either an evolution equation (in Stokes and potential flow limits) or thelong-time periodic drop response (for unsteady Stokes analysis). The analytical resultscompare satisfactorily with those obtained from the numerical simulation in Sarkar &Schowalter (2001), and the resonance characteristics are quantitatively explained. Thethree di erent solutions are compared with each other, and the results are presentedfor di erent parameters such as frequency, interfacial tension, viscosity ratio, densityratio and Reynolds number. Furthermore, the simple ODE model presented in theAppendix of Sarkar & Schowalter (2001) is shown to explain the asymptotic limitsof the present solution.

7 in Year 1993-Year 2000

The kinematics of a potential vortex offers an interesting flow history for a rheologically complex material, andearlier work on that subject led us to consider the behavior of a Newtonian drop in three related time dependentflow fields [K. Sarkar, W.R. Schowalter, Deformation of a two-dimensional drop at non-zero Reynolds number intime-periodic extensional flows: numerical simulation, J. Fluid Mech., 2000, submitted for publication; K. Sarkar,W.R. Schowalter, Deformation of a two-dimensional viscous drop in time-periodic extensional flows: analyticaltreatment, J. Fluid Mech., 2000, submitted for publication]. In the work reported here the drop, characterized by anupper-convected Maxwell model (UCM), is suspended in an incompressible Newtonian fluid. Again, three relatedflows are considered. The first is that of a potential vortex, modeled by an extensional flow field near the drop withrotating axes of stretching. The second is a generalization of the first and is calledrotating extensional(RE) flow,in which the frequency of revolution of the flow is varied independently of the shear rate. Finally, we consideroscillating extensional(OE) flow.Calculations were performed at small but non-zero Reynolds numbers using an ADI front-tracking/finite differ-ence method. We have developed an analytic elastic-viscous stress splitting scheme obtained by an integration byparts of the constitutive equation. The scheme explicitly separates the diffusive part of the momentum equation fora wide range of differential constitutive relations. An ADI implementation is executed for the diffusive part. Weinvestigate the effects of periodicity, Reynolds number and relaxation time on the drop dynamics. For a vortex andan RE flow, the long-time deformation reaches a steady value, and the drop attains a revolving, steady elliptic shape.The long-time values of deformation show complex non-monotonic behavior with variation in Weissenberg number,an effect of the decreased damping and increased elasticity, as well as the presence of a shear wave triggered by theUCM constitutive relation. The first two effects are modeled successfully by a simple ODE presented in AppendixA. The wave effects are briefly discussed in Appendix B.

This paper presents applications of boundary element methods to electrical impe- dance tomography. An algorithm for imaging the interior of a domain that consists of regions of constant conductivity is developed, that makes use of a simpler parametrization of the shapes of the regions to achieve efficiency. Numerical results from tests of this algorithm on synthetic data are presented, and show that the method is quite promising.

Ensemble averaging combined with multiple scattering ideas is applied to the Stokes flow over a stochastic rough surface. The surface roughness is modelled by compact protrusions on an underlying smooth surface. It is established that the effect of the roughness on the flow far from the boundary may be represented by replacing the no- slip condition on the exact boundary by a partial slip condition on the smooth surface. An approximate analysis is presented for a sparse distribution of arbitrarily shaped protrusions and explicit numerical results are given for hemispheres. Analogous conclusions for the two-dimensional case are obtained. It is shown that in certain cases a traction force develops on the surface at an angle with the direction of the flow.

Sarkar K, Prosperetti A 1995 “Effective boundary conditions for Laplace Equation with a Rough Boundary,” Proceedings of the Royal Society of London A, 451, 425-453.

A substantial amount of research on acoustic scattering by underwater bubbles is based on the theory of incoherent scattering. More recent work, devoted to much denser bubble assemblies, has instead used effective-media formulations that presuppose coherent effects. Here the mutual relationship between the two approaches is elucidated. It is shown that, underlying the incoherent results, is a WKB approximate solution of the effective equations. As an application, the scattering by tenuous subsurface bubble layers and acoustical bubble counting techniques are examined. Significant differences with previous results are found.

Sarkar K, Meneveau C 1993 “Gradients of potential fields on rough surface: perturbative calculation of the singularity distribution function f(a) for small surface,” Physical Review E, 47, 957-966.

This paper is a continuation of an earlier one [Prosperetti et al., J. Acoust. Soc. Am. 93, 3117-3127 (1993) ] in which the low-frequency backscattering of sound by hemispherical bubble clouds at the ocean's surface was studied. Here, clouds of various geometrical shapes (spheroids, spherical segments, cones, cylinders, ellipsoids) are considered and results in substantial agreement with the earlier ones and with the experiments of Chapman and Harris [J. Acoust. Soc. Am. 34, 1592-1597 ( 1962)] are found. The implication is that the backscattering levels are not strongly dependent on the shape of the clouds, which strengthens the earlier conclusion that bubble clouds produced by breaking waves can very well be responsible for the unexpectedly high backscattering levels observed experimentally. The accuracy of the Born approximation used by others for similar problems is also examined in the light of the exact results. Significant differences are found for gas concentrations by volume of the order of 0.01% or higher. Finally, shallow nonaxisymmetric plumes are briefly considered