Acoustic modification of collagen hydrogels facilitates cellular remodeling.

Developing tunable biomaterials that have the capacity to recreate the physical and biochemical characteristics of native extracellular matrices (ECMs) with spatial fidelity is important for a variety of biomedical, biological, and clinical applications. Several factors have made the ECM protein, collagen I, an attractive biomaterial, including its ease of isolation, low antigenicity and toxicity, and biodegradability. However, current collagen gel formulations fail to recapitulate the range of collagen structures observed in native tissues, presenting a significant challenge in achieving the full potential of collagen-based biomaterials. Collagen fiber structure can be manipulated in vitro through mechanical forces, environmental factors, or thermal mechanisms. Here, we describe a new ultrasound-based fabrication technology that exploits the ability of ultrasound to generate localized mechanical forces to control the collagen fiber microstructure non-invasively. The results indicate that exposing soluble collagen to ultrasound (7.8 or 8.8 MHz; 3.2-10 W/cm2) during hydrogel formation leads to local variations in collagen fiber structure and organization that support increased levels of cell migration. Furthermore, multiphoton imaging revealed increased cell-mediated collagen remodeling of ultrasound-exposed but not sham-exposed hydrogels, including formation of multicellular aggregates, collagen fiber bundle contraction, and increased binding of collagen hybridizing peptides. Skin explant cultures obtained from diabetic mice showed similar enhancement of cell-mediated remodeling of ultrasound-exposed but not sham-exposed collagen hydrogels. Using the mechanical forces associated with ultrasound to induce local changes in collagen fibril structure and organization to functionalize native biomaterials is a promising non-invasive and non-toxic technology for tissue engineering and regenerative medicine.

Low-intensity ultrasound increases endothelial cell nitric oxide synthase activity and nitric oxide synthesis.

Low-intensity ultrasound (US) increases tissue perfusion in ischemic muscle through a nitric oxide (NO)-dependent mechanism. We have developed a model to expose endothelial cells to well-characterized acoustic fields in vitro and investigate the physical and biological mechanisms involved. Human umbilical vein endothelial cells (HUVEC) or bovine aortic endothelial cells (BAEC) were grown in tissue culture plates suspended in a temperature-controlled water bath and exposed to US. Exposure to 27 kHz continuous wave US at 0.25 W cm(-2) for 10 min increased HUVEC media NO by 102 +/- 19% (P < 0.05) and BAEC by 117 +/- 23% (P < 0.01). Endothelial cell NO synthase activity increased by 27 +/- 24% in HUVEC and by 32 +/- 16% in BAEC (P < 0.05 for each). The cell response was rapid with a significant increase in NO synthesis by 10 s and a maximum increase after exposure for 1 min. By 30 min post-exposure NO synthesis declined to baseline, indicating that the response was transient. Unexpectedly, pulsing at a 10% duty cycle resulted in a 46% increase in NO synthesis over the response seen with continuous wave US, resulting in an increase of 147 +/- 18%. Cells responded to very low intensity US, with a significant increase at 0.075 W cm(-2) (P < 0.01) and a maximum response at 0.125 W cm(-2). US caused minor reversible changes in cell morphology but did not alter proliferative capacity, indicating absence of injury. We conclude that exposure of endothelial cells to low-intensity, low-frequency US increases NO synthase activity and NO production, which could be used to induce vasodilatation experimentally or therapeutically.

Bioeffects of positive and negative acoustic pressures in mice infused with microbubbles.

This study provided one test of the hypothesis that hemorrhage in tissues containing ultrasound (US) contrast agents results from inertial cavitation. The test relied on the prediction of classical cavitation theory that the response of microbubbles to negative pressures is much greater than it is for positive pressures. An endoscopic electrohydraulic lithotripter was used to generate a spherically diverging positive pressure pulse. A negative pressure pulse was produced by reflection of the positive pulse from a pressure release interface. Mice were injected with approximately 0. 1 mL of Albunex(R) and exposed to 100 pulses at either + 3.6 MPa or -3.6 MPa pressure amplitude. For comparison, mice were also exposed to the same acoustic fields without injection of contrast agents. Sham animals experienced the same protocols, with or without Albunex(R) injections, but were not exposed to the lithotripter fields. Following exposure, mice were scored for hemorrhage to various organs and tissues. When Albunex(R) was present in the vasculature, negative pressure pulses produced significantly more hemorrhage than positive pressures in tissues such as the kidney, intestine, skin, muscle, fat, mesentery and stomach.

The search for cavitation in vivo.

Until the mid 1970s, it was generally assumed that, with the short pulses of ultrasound (US) used in medical diagnosis, there was little need for concern about the possibility of inertial cavitation in vivo. This assumption came into question when experimental evidence indicated that killing of fruit fly larvae by diagnostically relevant US was associated with the presence of gas in the respiratory apparatus of the organisms. Independent theoretical contributions by Flynn and Apfel in the early 1980s made it clear that complacency in regard to cavitation was not warranted. Later, the mammalian lung, as with larva, was shown to be particularly vulnerable when it contained air. Yet, overall evidence suggests that lung hemorrhage is not consistent with the classical picture of inertial cavitation. Most recently, however, hemolysis and hemorrhage associated with the use of contrast agents have provided nearly incontrovertible evidence of the occurrence of cavitation in vivo.

Hemorrhage in murine fetuses exposed to pulsed ultrasound.

In the late-gestation fetal mouse, exposure to piezoelectric lithotripter fields at amplitudes < 1 MPa produced hemorrhages in tissues near developing bone, such as the head and limbs. This study was undertaken to determine if exposure to pulsed ultrasound at diagnostic frequencies produces similar hemorrhages in the late-gestation fetal mouse. On the 18th day of gestation, fetal mice were exposed in utero to pulsed ultrasound with a 10-micros pulse duration and 100-Hz pulse repetition frequency for a total exposure duration of 3 min. Hemorrhages occurred most often to the developing fetal head. At 1.2 MHz, a threshold for hemorrhage to the fetal head was determined at positive exposure pressures of approximately 4 MPa and corresponding negative pressures of approximately 2.5 MPa. The threshold increased with at least the first power of frequency.

Nonlinear propagation and the output indices.

By ignoring the effects of nonlinear propagation, current exposimetry protocols may yield significant underestimates of the acoustic pressure in situ. This problem can be avoided simply by (1) extrapolating pressures linearly from low amplitude measurements in water and (2) linearly derating these values to obtain estimates of fields in situ. The mechanical index was designed to provide an indication of temporal peak acoustic fields for use in prediction of nonthermal biological effects in tissues. At low outputs, the mechanical index, together with the frequency, gives the peak negative pressure near the focus of the field. As currently formulated, however, the pressure used in the mechanical index may be far from the focus at high output levels. Recommendations of the World Federation of Ultrasound in Medicine and Biology avoid the underestimate associated with nonlinear propagation as well as other problems with the mechanical index and may be preferable in dealing with non-thermal bioeffects. The thermal indices that are implemented currently in the Output Display Standard (American Institute of Ultrasound in Medicine/National Electrical Manufacturers' Association) are affected less seriously by nonlinear propagation.

Enhancement of fibrinolysis with 40-kHz ultrasound.

BACKGROUND: Ultrasound at frequencies of 0.5 to 1 MHz and intensities of > or =0.5 W/cm2 accelerates enzymatic fibrinolysis in vitro and in some animal models, but unacceptable tissue heating can occur, and limited penetration would restrict application to superficial vessels. Tissue heating is less and penetration better at lower frequencies, but little information is available regarding the effect of lower-frequency ultrasound on enzymatic fibrinolysis. We therefore examined the effect of 40-kHz ultrasound on fibrinolysis, tissue penetration, and heating. METHODS AND RESULTS: 125I-fibrin-radiolabeled plasma clots in thin-walled tubes were overlaid with plasma containing tissue plasminogen activator (tPA) and exposed to ultrasound. Enzymatic fibrinolysis was measured as solubilization of radiolabel. Tissue attenuation and heating were examined in samples of porcine rib cage. Fibrinolysis was increased significantly in the presence of 40-kHz ultrasound at 0.25 W/cm2, reaching 39+/-7% and 93+/-11% at 60 minutes and 120 minutes, compared with 13+/-8% and 37+/-4% in the absence of ultrasound (P<0.0001). The acceleration of fibrinolysis increased at higher intensities. Attenuation of the ultrasound field was only 1.7+/-0.5 dB/cm through the intercostal space and 3.4+/-0.9 dB/cm through rib. Temperature increments in rib were <1 C/(W/cm2). CONCLUSIONS: These findings indicate that 40-kHz ultrasound significantly accelerates enzymatic fibrinolysis at intensities of > or =0.25 W/cm2 with excellent tissue penetration and minimal heating. Externally applied 40-kHz ultrasound at low intensities is a potentially useful therapeutic adjunct to enzymatic fibrinolysis with sufficient tissue penetration for both peripheral vascular and coronary applications.

Thresholds for premature contractions in murine hearts exposed to pulsed ultrasound.

A single pulse of high intensity ultrasound can produce either a premature ventricular contraction or a reduction in the aortic pressure in frog hearts. The objective of this study was to determine whether similar ultrasound exposures can produce premature contractions in the mammalian heart. The cardiac activity of murine hearts in vivo was monitored noninvasively using electrocardiography and plethysmography. Each ultrasound exposure was a single pulse of ultrasound, several milliseconds in duration, delivered to the murine heart during diastole. The thresholds for producing a premature contraction with a 5-ms ultrasound pulse at 1.2 MHz was approximately 2 MPa peak positive pressure. The occurrence of premature contractions decreased as the duration of the ultrasound pulse decreased. These results found with the mammalian heart are similar to those reported earlier for the frog heart. No damage to cardiac tissue was observed grossly, although significant hemorrhage occurred to adjacent lung tissue.

Age dependence of ultrasonically induced lung hemorrhage in mice.

Thresholds for ultrasonically induced lung hemorrhage were determined in neonatal mice (24-36 h old), juvenile mice (14 d old) and adult mice (8-10 weeks old) to assess whether or not the threshold for lung hemorrhage is dependent upon age. Ultrasonic exposures were at 1.15 MHz with a pulse length of 10 microseconds, pulse repetition frequency of 100 Hz and a total exposure duration of 3 min. The threshold for lung hemorrhage occurred at a peak positive acoustic pressure of approximately 1 MPa for mice in all three age groups. Although the thresholds were similar for neonatal, juvenile and adult mice, the sizes of the suprathreshold hemorrhages were significantly larger in adult mice than in neonatal or juvenile mice.

Ultrasonically induced lung hemorrhage in young swine.

Ten-day old swine were used in the final step of a study of the age dependence of the threshold for lung hemorrhage resulting from exposure to diagnostically relevant levels of pulsed ultrasound. A 2.3-MHz focused transducer (pulse length of 10 microseconds, 100-Hz pulse repetition frequency) was incremented vertically at several sites for a distance of 2 or 2.5 cm over the chest of the subject for a total exposure period of 16 or 20 min. The procedure was repeated at a total of four sites per animal. Animals were euthanized and lungs were scored by visual inspection for numbers and areas of gross hemorrhages. The threshold level for hemorrhage was approximately 1.3-MPa peak positive pressure in water and the surface of the animal or, at the surface of the lung, 0.8-MPa peak positive pressure, 0.8-MPa fundamental pressure, 0.7-MPa maximum negative pressure and 20 Wcm-2 pulse average intensity. These values are essentially the same as those reported previously for neonatal swine, and neonatal, juvenile and adult mice.

Effects of pulsed ultrasound on the frog heart: III. The radiation force mechanism.

Earlier studies have shown that a single, millisecond duration pulse of ultrasound delivered to the frog heart in vivo during systole can produce a reduction in the developed aortic pressure, while a pulse delivered during diastole can produce a premature ventricular contraction. The threshold for these effects is 5-10 MPa with a 5-ms pulse. Since cardiac tissues respond to mechanical stimulation, the objective of this study was to investigate acoustic radiation force as a possible mechanism for the observed effects of ultrasound on the frog heart. In two experiments, the radiation force exerted on the heart was varied by varying the ultrasonic frequency and the acoustic beam width. Results of these studies indicated that the rate of occurrence of the reduced aortic pressure effect was directly correlated with the magnitude of the radiation force exerted on the heart. A third experiment tested the radiation force mechanism directly by placing an acoustic reflector on the frog heart. The acoustic reflector maximized the radiation force delivered to the heart, but eliminated direct interaction of the ultrasound with the heart and experimentally eliminated heating and cavitation as mechanisms of action. The reduced aortic pressure effect was observed with the reflector on the heart, indicating that radiation force is capable of producing this effect. No premature ventricular contractions were observed with the acoustic reflector over the heart, suggesting that another property of the exposure may be responsible for this bioeffect.

Thresholds for fetal hemorrhages produced by a piezoelectric lithotripter.

Hemorrhage to fetal tissues occurred when late-term pregnant mice were exposed to lithotripter fields of relatively low amplitude. These hemorrhages were always observed in tissues near developing bone or cartilaginous structures such as the head, limbs and ribs, while soft tissues distant from bone were relatively free of hemorrhage. Thresholds for hemorrhage in the fetus were determined for exposures of pregnant mice on the 18th day of gestation to 200 pulses from a piezoelectric lithotripter. Animals were exposed to axial peak positive pressures of either 0 (sham), 1, 2, 3, 5 or 10 MPa. Thresholds for hemorrhage to the head, limbs, ribs and lung were all < 1 MPa.

Hemolysis in vivo from exposure to pulsed ultrasound.

Ultrasonically induced hemolysis in vivo when a commercial ultrasound contrast agent, Albunex, was present in the blood. Murine hearts were exposed for 5 min at either 1.15 or 2.35 MHz with a pulse length of 10 microseconds and pulse repetition frequency of 100 Hz. During the exposure period, four boluses of Albunex were injected into a tail vein for a total of approximately 0.1 mL of Albunex. Following exposure, blood was collected by heart puncture and centrifuged, and the plasma was analyzed for hemoglobin concentration. With Albunex present in the blood, the threshold for hemolysis at 1.15 MHz was 3.0 +/- 0.8 MPa (mean +/- SD) peak positive pressure (approximately 1.9 MPa negative pressure, approximately 180 W cm-2 pulse average intensity). For the highest exposure levels (10 MPa peak positive pressure at the surface of the animal), the mean value for hemolysis was approximately 4% at 1.15 MHz and 0.46% at 2.35 MHz, i.e., the threshold at 2.35 MHz is > 10 MPa peak positive pressure. In contrast, hemolysis in control mice receiving saline injections at 10 MPa or sham-exposed (0 MPa) mice receiving Albunex was approximately 0.4%.

Remnants of Albunex nucleate acoustic cavitation.

Mice were injected with 0.1 mL Albunex and exposed to 200 pulses from a piezoelectric lithotripter at times ranging from 5 min to 24 h following injection. Each pulse was approximately 1.5 sinusoidal oscillations at a fundamental frequency of approximately 0.1 MHz with pressure amplitude of approximately 2 MPa. Although the contrast agent ceases to be an effective scatterer of diagnostic ultrasound after a few minutes in the circulation, the modest lithotripter exposures caused significant hemorrhaging in bladder, mesentery and intestine for periods of up to 4 h after injection. The results demonstrate either that highly stable bubbles much smaller than resonance size or air-containing fragments of the shells of Albunex serve as effective nuclei for acoustic cavitation.

The influence of contrast agents on hemorrhage produced by lithotripter fields.

Ultrasonic contrast agents greatly increase the side effects of low-amplitude lithotripter fields in mice. Using a piezoelectric lithotripter, adult mice were exposed to 200 lithotripter pulses with a peak positive pressure amplitude of 2 MPa. During the exposure period, mice were injected with approximately 0.1 mL of the ultrasonic contrast agent Albunex. For comparison, another group of mice experienced the same lithotripter exposures, but were not injected with contrast agent. Following exposures, animals were sacrificed and observed for hemorrhage in various organs and tissues. Mice exposed to the lithotripter field alone had minimal hemorrhage only in the intestine and lung. In comparison, mice injected with Albunex during exposure exhibited extensive hemorrhage in the intestine, kidney, muscle, mesentery, stomach, bladder, seminal vesicle and fat.

Albunex Does Not Increase the Sensitivity of the Lung to Pulsed Ultrasound.

If cavitation in the vasculature of the lung is the physical mechanism responsible for lung hemorrhage, then addition of cavitation nuclei to the blood should enhance the bioeffect. To test the cavitation hypothesis, the extent of lung hemorrhage in mice injected with the echocontrast agent, Albunex(R), was compared to lung hemorrhage in animals injected with saline. Animals were exposed for 5 minutes to 1.1-MHz pulsed ultrasound (10 µs pulse length, 100-Hz pulse repetition frequency) at a peak positive pressure at the surface of the animal of 2 MPa. This exposure is approximately twice the threshold pressure amplitude for lung hemorrhage. Lesion areas did not differ significantly in the two groups of animals and were approximately equal to the lesion area in uninjected mice from an earlier study where acoustic exposures were the same. Neither this study nor a related study of hemolysis in vivo suggests that use of Albunex in echocardiographic procedures increases the risk of bioeffects.

Bioeffects of positive and negative acoustic pressures in vivo.

In water, the inertial collapse of a bubble is more violent after expansion by a negative acoustic pressure pulse than when directly compressed by a positive pulse of equal amplitude and duration. In tissues, gas bodies may be limited in their ability to expand and, therefore, the relatively strong effectiveness of negative pressure excursions may be tempered. To determine the relative effectiveness of positive and negative pressure pulses in vivo, the mortality rate of Drosophila larvae was determined as a function of exposure to microsecond length, nearly unipolar, positive and negative pressure pulses. Air-filled tracheae in the larvae serve as biological models of small, constrained bubbles. Death from exposure to ultrasound has previously been correlated with the presence of air in the respiratory system. The degree of hemorrhage in murine lung was also compared using positive and negative pulses. The high sensitivity of lung to exposure to ultrasound also depends on its gas content. The mammalian lung is much more complex than the respiratory system of insect larvae and, at the present time, it is not clear that acoustic cavitation is the physical mechanism for hemorrhage. A spark from an electrohydraulic lithotripter was used to produce a spherically diverging positive pulse. An isolated negative pulse was generated by reflection of the lithotripter pulse from a pressure release interface. Pulse amplitudes ranging from 1 to 5 MPa were obtained by changing the proximity of the source to the biological target. For both biological effects, the positive pulse was found to be at least as damaging as the negative pulse at comparable temporal peak pressure levels. These observations may be relevant to an evaluation of the mechanical index (MI) as an exposure parameter for tissues including lung since MI currently is defined in terms of the magnitude of the negative pressure in the ultrasound field.

A test for cavitation as a mechanism for intestinal hemorrhage in mice exposed to a piezoelectric lithotripter.

This study tested the hypothesis that intestinal hemorrhage produced by exposure to lithotripter fields depends upon the presence of gas in the intestine. The extent of hemorrhage in the gas-containing intestines of pregnant mice was compared to the amount of hemorrhage in the bubble-free intestines of their fetuses. On day 18 of gestation, the abdominal regions of pregnant C3H mice (n = 6) were exposed to 200 pulses from a piezoelectric lithotripter. Acoustic pulses had a peak pressure amplitude of 10 MPa and were administered at a rate of approximately 1 Hz. All maternal intestines showed hemorrhagic regions extending several centimeters in length. In contrast, only 1 of 43 exposed fetuses showed an intestinal hemorrhage and this one lesion was less than 1 mm in diameter. These results support the hypothesis of the study and are consistent with a cavitation-related mechanism for the production of intestinal hemorrhage by exposure to acoustic fields.

Thresholds for ultrasonically induced lung hemorrhage in neonatal swine.

The threshold for generation of lung hemorrhage in adult mice by pulsed ultrasound has been shown to be approximately 1 MPa at the surface of the lung (10-microseconds pulse and a carrier frequency of 2 MHz). This investigation used neonatal swine to determine if the findings for mice can be generalized to other species. After exploratory observations, the inverse sampling method was used in a primary study (22 animals, 88 exposure sites) to determine the threshold for lung hemorrhage in neonatal swine. The primary study was followed by a separate confirmation study (13 animals, 48 exposure sites), testing the conclusions of the first study and comparing damage at subthreshold levels with sham-exposed animals. A separate investigation explored the histological nature of tissue damage at suprathreshold levels. A 2.3-MHz focused transducer (10 microseconds at 100-Hz pulse-repetition frequency) was incremented vertically for a distance of 2 cm over the chest of the subject for a total exposure period of 16 min. Animals were euthanized and lungs were scored by visual inspection for numbers and areas of gross hemorrhages. The threshold level for hemorrhage was approximately 1.5 MPa peak positive pressure in water at the surface of the animal or, at the surface of the lung, 1.1 MPa peak positive pressure, 1 MPa fundamental pressure, 0.9 MPa maximum negative pressure, 25 W cm-2 pulse average intensity or a mechanical index of 0.6. These values are essentially the same as those reported for adult mice.

Exposure-time dependence of the threshold for ultrasonically induced murine lung hemorrhage.

Although the extent of suprathreshold damage to murine lung that results from exposure to pulsed ultrasound increases with time, the threshold level for lung hemorrhage is relatively insensitive to total exposure time. Adult mice were exposed for 20 s and 3 min to 2.3-MHz ultrasound (10-microseconds pulses, 100-Hz pulse repetition frequency) at peak positive pressures ranging up to 3 MPa. Threshold pressures for the two exposure times, 1.6 MPa and 1.4 MPa, respectively, are the same within the statistical significance of the measurements.