High-frequency ultrasonic imaging of growth and development in manufactured engineered oral mucosal tissue surfaces.

This study uses high-resolution ultrasound to examine the growth and development of engineered oral mucosal tissues manufactured under aseptic conditions. The specimens are a commercially available natural tissue scaffold, AlloDerm, and oral keratinocytes seeded onto AlloDerm to form an ex vivo-produced oral mucosal equivalent (EVPOME) suitable for intra-oral grafting. The seeded cells produce a keratinized protective upper layer that smooths out any remaining surface irregularities on the underlying AlloDerm. Two-dimensional acoustic imaging of unseeded AlloDerm and developing EVPOMEs was performed on each day of their growth and development, each tissue specimen being imaged under aseptic conditions (total time from seeding to maturation: 11 d). Ultrasonic monitoring offers us the ability to determine the constituents of the EVPOME that are responsible for changes in its mechanical behavior during the manufacturing process. Ultrasonic monitoring affords us an opportunity to non-invasively assess, in real time, tissue-engineered constructs before release for use in patient care.

Using an ultrasound elasticity microscope to map three-dimensional strain in a porcine cornea.

An ultrasound elasticity microscope was used to map 3-D strain volume in an ex vivo porcine cornea to illustrate its ability to measure the mechanical properties of this tissue. Mechanical properties of the cornea play an important role in its function and, therefore, also in ophthalmic diseases such as kerataconus and corneal ectasia. The ultrasound elasticity microscope combines a tightly focused high-frequency transducer with confocal scanning to produce high-quality speckle over the entire volume of tissue. This system and the analysis were able to generate volume maps of compressional strain in all three directions for porcine corneal tissue, more information than any previous study has reported. Strain volume maps indicated features of the cornea and mechanical behavior as expected. These results constitute a step toward better understanding of corneal mechanics and better treatment of corneal diseases.

Characterizing morphology and nonlinear elastic properties of normal and thermally stressed engineered oral mucosal tissues using scanning acoustic microscopy.

This study examines the use of high-resolution ultrasound to monitor changes in the morphology and nonlinear elastic properties of engineered oral mucosal tissues under normal and thermally stressed culture conditions. Nonlinear elastic properties were determined by first developing strain maps from acoustic ultrasound, followed by fitting of nonlinear stress-strain data to a 1-term Ogden model. Testing examined a clinically developed ex vivo produced oral mucosa equivalent (EVPOME). As seeded cells proliferate on an EVPOME surface, they produce a keratinized protective upper layer that fills in and smoothens out surface irregularities. These transformations can also alter the nonlinear stress/strain parameters as EVPOME cells differentiate. This EVPOME behavior is similar to those of natural oral mucosal tissues and in contrast to an unseeded scaffold. If ultrasonic monitoring could be developed, then tissue cultivation could be adjusted in-process to account for biological variations in their development of the stratified cellular layer. In addition to ultrasonic testing, an in-house-built compression system capable of accurate measurements on small (∼1.0-1.5 cm(2)) tissue samples is presented. Results showed a near 2.5-fold difference in the stiffness properties between the unstressed EVPOME and the noncell-seeded acellular scaffold (AlloDerm(®)). There were also 4×greater differences in root mean square values of the thickness in the unseeded AlloDerm compared to the mature unstressed EVPOME; this is a strong indicator for quantifying surface roughness.

Comparison of scanning acoustic microscopy and histology images in characterizing surface irregularities among engineered human oral mucosal tissues.

Acoustic microscopy was used to monitor an ex vivo produced oral mucosal equivalent (EVPOME) developed on acellular cadaveric dermis (AlloDerm®). As seeded cells adhered and grew, they filled in and smoothed out the surface irregularities, followed by the production of a keratinized protective outermost layer. If noninvasive in vitro ultrasonic monitoring of these cellular changes could be developed, then tissue cultivation could be adjusted in-process to account for biologic variations in the development of these stratified cell layers. Cultured keratinocytes (from freshly obtained oral mucosa) were harvested and seeded onto AlloDerm® coated with human type IV collagen and cultured 11 days. EVPOMEs were imaged on the 11th day post-seeding using a scanning acoustic microscope (SAM) that consists of a single-element transducer: 61 MHz center frequency, 32 MHz bandwidth, 1.52 f-number. The specimen surface was determined by thresholding the magnitude of the signal at the first axial incidence of a value safely above noise: 20-40 dB above the signal for the water and 2-dimensional (2-D) ultrasonic images were created using confocal image reconstruction. A known area from each micrograph was divided into 12-40 even segments and examined for surface irregularities. These irregularities were quantified and one-way analysis of variance (ANOVA) and linear regression analysis were performed to correlate the surface profiles for both the AlloDerm® and EVPOME specimens imaged by SAM. Histology micrographs of the AlloDerm® and EVPOME specimens were also prepared and examined for surface irregularities. Unseeded AlloDerm® averaged seven to nine surface changes per 400 µm. The number of changes in surface irregularities decreased to two to three per 400 µm on the mature EVPOMEs. The numbers of surface irregularities between the unseeded AlloDerm® vs. developing EVPOME are similar for both histology and SAM 2-D B-scan images. For the EVPOME 2-D B-scan micrographs produced by SAM, the decrease in surface irregularities is indicative of the stratified epithelium formed by seeded oral keratinocytes; verified in the histology images between the AlloDerm® and EVPOME. A near 1:1 linear correlation shows the similarities between the two imaging modalities. SAM demonstrates its ability to discern the cell development and differentiation occurring on the EVPOME devices. Unlike histology, SAM measurements are noninvasive and can be used to monitor tissue graft development without damaging any cells/tissues.

Acoustic microscopy analyses to determine good vs. failed tissue engineered oral mucosa under normal or thermally stressed culture conditions.

This study uses scanning acoustic microscopy (SAM) ultrasonic profilometry to determine acceptable vs. failed tissue engineered oral mucosa. Specifically, ex vivo-produced oral mucosal equivalents (EVPOMEs) under normal or thermally stressed culture conditions were scanned with the SAM operator blinded to the culture conditions. As seeded cells proliferate, they fill in and smooth out the surface irregularities; they then stratify and produce a keratinized protective upper layer. Some of these transformations could alter backscatter of ultrasonic signals and in the case of the thermally stressed cells, produce backscatter similar to an unseeded device. If non-invasive ultrasonic monitoring could be developed, then tissue cultivation could be adjusted to measure biological variations in the stratified surface. To create an EVPOME device, oral mucosa keratinocytes were seeded onto acellular cadaveric dermis. Two sets of EVPOMEs were cultured: one at physiological temperature 37 °C and the other at 43 °C. The specimens were imaged with SAM consisting of a single-element transducer: 61 MHz center frequency, 32 MHz bandwidth, 1.52 f#. Profilometry for the stressed and unseeded specimens showed higher surface irregularities compared to unstressed specimens. Elevated thermal stress retards cellular differentiation, increasing root mean square values; these results show that SAM can potentially monitor cell/tissue development.

Three-dimensional mapping of strain in ex vivo porcine cornea with an ultrasound elasticity microscope.

High frequency strain mapping of a porcine cornea was produced by three-dimensional speckle tracking of a three-dimensional confocally merged ultrasonic data set. Previous two-dimensional elasticity imaging was limited by speckle moving in the non-imaged dimension. This study used an ultrasonic transducer (53 MHz center frequency, 31 MHz bandwidth, 1.67 f#) scanned in three dimensions. A fresh porcine eye globe was embedded in gelatin up to the cornea/sclera junction and pressurized to physiological pressure. A portion of the cornea was imaged with a single element transducer scanned in three directions. Three-dimensional volume sets were created from the data by confocally merging volume sets at several depths. In the vertical dimension, parallel to ultrasonic propagation, the transducer was moved by nearly half its depth of field. Overlaps between adjacent depths were correlated using phase-sensitive speckle tracking to determine precise shifts. Two-dimensional images over multiple depths of field were combined to form a single 2-D image over an entire vertical scan range. Multiple planes were then stacked in the remaining direction to form a 3-D volume set. Next, the cornea was deformed and then imaged again. Three-dimensional speckle tracking was used to create strain maps from the two volume sets. Vertical strain, horizontal strain, and transverse strain behave as expected and provide insight into mechanical properties of corneal tissue.

Non-linear stress-strain measurements of ex vivo produced oral mucosal equivalent (EVPOME) compared to normal oral mucosal and skin tissue.

Stress-strain curves of oral mucosal tissues were measured using direct mechanical testing. Measurements were conducted on both natural oral mucosal tissues and engineered devices, specifically a clinically developed ex vivo produced oral mucosal equivalent (EVPOME). As seeded cells proliferate on EVPOME devices, they produce a keratinized protective upper layer which fills in surface irregularities. These transformations can further alter stress-strain parameters as cells in EVPOME differentiate, more similar to natural oral mucosal tissues in contrast to an unseeded scaffold. In addition to tissue devices grown under normal conditions (37 °C), EVPOMEs were also produced at 43 °C. These thermally stressed specimens model possible failure mechanisms. Results from a mechanical deformation system capable of accurate measurements on small (approximately 1.0-1.5 cm(2)) cylindrical tissue samples are presented. Deformations are produced by lowering a circular piston, with a radius smaller than the sample radius, onto the center of the sample. Resulting force is measured with a precision electronic balance. Cultured EVPOME was less stiff than AlloDerm®, but similar to native porcine buccal tissue. Porcine skin and porcine palate tissues were even less stiff. Thermally stressed EVPOME was less stiff than normally cultured EVPOME as expected because stressed keratin cells were damaged reducing the structural integrity of the tissue.

Mapping elasticity in human lenses using bubble-based acoustic radiation force.

This study uses acoustic radiation pressure to displace a femtosecond laser-produced bubble in human lens tissue. Bubble displacement is monitored with low-amplitude, high-resolution ultrasound. Displacements are compensated by bubble size determined from ultrasonic backscatter. The Young's modulus is proportional to the inverse of the compensated displacement with the constant of proportionality determined from similar measurements in a controlled gelatin sample. Multiple measurements were obtained on 12 human lens specimens grouped into two age categories, middle-age (about 40 years old) and old-age (63-70 years old). There were 3 lenses from 2 donors in the middle-age group and 9 lenses from 5 donors in the old-age group. At each radial position, the median value was computed for all measurements within each group. For middle-age lenses, Young's modulus ranged from 5.2kPa in the center to 1.1kPa on the periphery. For old-age lenses, Young's modulus ranged from 10.6kPa in the center to 1.4kPa on the periphery. These values are the same order of magnitude as previous measurements using other techniques. The age related change in elasticity distribution is also similar to a previous study. Radially varying elasticity may provide insight into the mechanics of accommodation.

Bubble-based acoustic radiation force using chirp insonation to reduce standing wave effects.

Bubble-based acoustic radiation force can measure local viscoelastic properties of tissue. High intensity acoustic waves applied to laser-generated bubbles induce displacements inversely proportional to local Young's modulus. In certain instances, long pulse durations are desirable but are susceptible to standing wave artifacts, which corrupt displacement measurements. Chirp pulse acoustic radiation force was investigated as a method to reduce standing wave artifacts. Chirp pulses with linear frequency sweep magnitudes of 100, 200 and 300 kHz centered around 1.5 MHz were applied to glass beads within gelatin phantoms and laser-generated bubbles within porcine lenses. The ultrasound transducer was translated axially to vary standing wave conditions, while comparing displacements using chirp pulses and 1.5 MHz tone burst pulses of the same duration and peak rarefactional pressure. Results demonstrated significant reduction in standing wave effects using chirp pulses, with displacement proportional to acoustic intensity and bubble size.

Mapping age-related elasticity changes in porcine lenses using bubble-based acoustic radiation force.

Bubble-based acoustic radiation force aims to measure highly localized tissue viscoelastic properties. In the current investigation, acoustic radiation force was applied to laser-induced bubbles to measure age-related changes in the spatial distribution of elastic properties within in vitro porcine lenses. A potential in vivo technique to map lens elasticity is crucial to understanding the onset of presbyopia and develop new treatment options. Bubble-based acoustic radiation force was investigated as a technique to measure the spatial elasticity distribution of the lens in its natural state without disrupting the lens capsule. Laser-induced optical breakdown (LIOB) generated microbubbles in a straight line across the equatorial plane of explanted porcine lenses with 1mm lateral spacing. Optical breakdown occurs when sufficiently high threshold fluence is attained at the focus of femtosecond pulsed lasers, inducing plasma formation and bubble generation. A two-element confocal ultrasonic transducer applied 6.5 ms acoustic radiation force-chirp bursts with the 1.5 MHz outer element while monitoring bubble position within the lens using pulse-echoes with the 7.44 MHz inner element. A cross-correlation method was used to measure bubble displacements and determine exponential time constants of the temporal responses. Maximum bubble displacements are inversely proportional to the local Young's modulus, while time constants are indicative of viscoelastic properties. The apparent spatial elasticity distributions in 41 porcine lenses, ranging from 4 months to 5 years in age, were measured using bubble-based acoustic radiation force. Bubble displacements decrease closer to the porcine lens center, suggesting that the nucleus is stiffer than the cortex. Bubble displacements decrease with increasing lens age, suggesting that porcine lenses become stiffer with age. Bubble-based acoustic radiation force may be well-suited as a potential in vivo technique to spatially map elastic properties of the lens and guide therapeutic procedures aimed at restoring accommodation.

Bubble-based acoustic radiation force elasticity imaging.

Acoustic radiation force is applied to bubbles generated by laser-induced optical breakdown (LIOB) to study viscoelastic properties of the surrounding medium. In this investigation, femtosecond laser pulses are focused in the volume of gelatin phantoms of different concentrations to form bubbles. A two-element confocal ultrasonic transducer generates acoustic radiation force on individual bubbles while monitoring their displacement within a viscoelastic medium. Tone burst pushes of varying duration have been applied by the outer element at 1.5 MHz. The inner element receives pulse-echo recordings at 7.44 MHz before, during, and after the excitation bursts, and crosscorrelation processing is performed offline to monitor bubble position. Maximum bubble displacements are inversely related to the Young's moduli for different gel phantoms, with a maximum bubble displacement of over 200 microm in a gel phantom with a Young's modulus of 1.7 kPa. Bubble displacements scale with the applied acoustic radiation force and displacements can be normalized to correct for differences in bubble size. Exponential time constants for bubble displacement curves are independent of bubble radius and follow a decreasing trend with the Young's modulus of the surrounding medium. These results demonstrate the potential for bubble-based acoustic radiation force methods to measure tissue viscoelastic properties.

Acoustic detection of controlled laser-induced microbubble creation in gelatin.

A high-frequency (85 MHz) acoustic technique is used to identify system parameters for controlled laser-induced microbubble creation inside tissue-mimicking, gelatin phantoms. Microbubbles are generated at the focus of an ultrafast 793-nm laser source and simultaneously monitored through ultrasonic pulse-echo recordings. Displayed in wavefield form, these recordings illustrate microbubble creation, and integrated backscatter plots provide specifics about microbubble characteristics and dissolution behavior. By varying laser parameters, including pulse fluence (or pulse energy flux, J/cm2), total number of pulses delivered, and the period between pulses, the size, lifetime, and dissolution dynamics of laser-induced microbubbles may be independently controlled. Pulse fluence is the main size-controlling parameter, whereas both increases in pulse fluence and pulse number can lengthen microbubble lifetime from tens to hundreds of milliseconds. In short, a microbubble of particular lifetime does not necessarily have to be of a particular size. Microbubble behavior, furthermore, is independent of pulse periods below a fluence-dependent threshold value, but it exhibits stochastic behavior if pulse repetition is too slow. These results demonstrate that laser pulse fluence, number, and period may be varied to deposit energy in a specific temporal manner, creating and stabilizing microbubbles with particular characteristics and, therefore, potential uses in sensitive acoustic detection and manipulation schemes.

Trapping cavitation bubbles with a self-focused laser beam.

We observed that laser-induced cavitation bubbles in water can be trapped in a self-focused laser beam. Both optical imaging and acoustic detection have been utilized to confirm bubble trapping. Transverse and longitudinal trapping forces were measured to be as large as 87 and 11 pN, respectively. This result is contrary to conventional wisdom, since the mechanism of trapping in conventional optical tweezers implies that a low-index particle (a bubble being the limiting case) should be antitrapped.

Strain imaging of corneal tissue with an ultrasound elasticity microscope.

PURPOSE: We hypothesize that high-resolution elasticity measurements can guide corrective refractive surgery of the cornea. Elasticity measurements would improve surgical outcomes by adding biomechanical information not used in existing clinical nomograms. As an initial investigation, we determined the usefulness and evaluated the ability of our ultrasound elasticity microscope by measuring strain ex vivo in an intact porcine eye globe. METHODS: Strain was predicted with a finite element model guided by direct mechanical measurements of corneal elasticity. Next, a porcine cornea was deformed with a slitted plate while being imaged with ultrasound. For high spatial resolution, the ultrasound elasticity microscope uses a 50 MHz transducer with a 1.4 f/number. It produces high-quality conventional ultrasonic B-scans over large thicknesses by confocal processing. Strain was calculated from tracking speckle in these images after deformation. This technique is compatible with in vivo measurements. RESULTS: Compressional and expansional deformations were the same order of magnitude from -3.5% to as great as +3.5%. Strain imaging indicated the stroma expanded into the slit of the deformation plate while Bowman's layer compressed. This bipolar variation within a specimen is unusual. Within the stroma, a variation of strain with depth was measured suggesting a distribution of elasticity. Results compared favorably with the finite element model. CONCLUSION: An ultrasound elasticity microscope can produce high-resolution strain images throughout the corneal depth. Various layers with different elastic properties appeared as different strains in the images.