氧化铝与冷冻保护剂浓度对 PVA 剪切波黏弹性体模声学与黏弹特性的影响
Characterization of tissue-mimicking polyvinyl alcohol phantoms for shear wave viscoelastography: effects of aluminum oxide and cryoprotectant concentration
一项研究系统评估了氧化铝及冷冻保护剂乙二醇（EG）和丙二醇（PG）浓度对 10%（w/v）PVA 组织模拟体模声学与黏弹特性的影响。氧化铝浓度从 0% 增至 10%（w/v）时衰减从 0.12 升至 0.54 dB/cm/MHz（r=0.98），纵波声速（1576–1583 m/s）与黏度（1.58–2.15 Pa·s）无显著变化，但剪切模量随浓度升高而增大。
来源：Frontiers in Acoustics 最新文章 · CC BY 4.0 · 原文 · 许可条款
Abstract
Introduction:
Polyvinyl alcohol (PVA) phantoms are under development for quality control and evaluation of shear wave elastography. These are typically formulated with aluminum oxide to mimic soft-tissue attenuation. Further, cryoprotectants, such as ethylene glycol (EG) and propylene glycol (PG), suppress ice crystal formation during freeze-thaw cycling and improve homogeneity. This study systematically assesses the effects of aluminum oxide and the cryoprotectants EG and PG on the acoustic and viscoelastic properties of PVA phantoms for elastography applications.
Methods:
Phantoms were fabricated from 10% (w/v) PVA, with aluminum oxide varied from 0%–10% (w/v), and with EG or PG varied from 0%–20% (v/v). Microstructure was examined by scanning electron microscopy. Density, longitudinal sound speed, and attenuation were measured for each phantom (n = 5 each). Shear-wave viscoelastography was performed to estimate shear modulus and viscosity.
Results:
Longitudinal sound speed (1576–1583 m/s) and viscosity (1.58–2.15 Pa s) did not change significantly with increasing aluminum oxide concentration. On the other hand, attenuation increased from 0.12–0.54 dB/cm/MHz (r = 0.98; p < 0.001), density rose from 1.03–1.14 g/cm3 (r = 0.91, p < 0.001), and shear modulus increased from 3.05 to 5.32 kPa (r = 0.79; p < 0.001). Longitudinal sound speed and attenuation measurements increased with increasing cryoprotectant concentration. Sound speed measurements spanned 1535–1643 m/s for EG (r = 0.99, p < 0.001) and 1535–1666 m/s for PG (r = 0.99; p < 0.001). Attenuation spanned 0.27–0.40 dB/cm/MHz for EG (r = 0.84; p < 0.001) and 0.27–0.39 dB/cm/MHz for PG (r = 0.82; p < 0.001). Shear modulus estimates changed non-monotonically with cryoprotectant concentration, decreasing from 6.94 kPa at 0% EG to 3.81 kPa at 15%, and increasing to 8.01 kPa at 20%. The shear modulus decreased from 0% to 10% PG and remained constant from 10% to 20%. Viscosity decreased from 0%–5% but was not significantly different between 5%-20% for both EG and PG phantoms.
Discussion:
Aluminum oxide doping simultaneously increases attenuation and alters viscoelastic properties, while cryoprotectants drive nonlinear shifts in mechanical properties. These results demonstrate how the additives concomitantly influence the acoustic and viscoelastic properties of PVA phantoms. These findings support efforts to standardize phantom fabrication protocols and control confounding variables.
1 Introduction
Ultrasound tissue-mimicking phantoms are commonly used for equipment calibration and technique verification, enabling the evaluation of imaging methods and reconstruction algorithms reproducibly before they are applied in clinical settings (; ). Phantoms are fabricated with acoustic and mechanical properties that replicate those of soft tissues (; ). The longitudinal sound speed and attenuation are two relevant acoustic parameters, and the mechanical properties of phantoms are tuned for elastography applications. Most phantoms that were used to verify new techniques, such as those of shear wave elastography, were assumed to be purely elastic. However, recent studies have shown that assuming inherently viscoelastic materials, e.g., biological tissues, as elastic introduces substantial errors in modulus estimates (; ; ). Therefore, shear wave viscoelasticity imaging, i.e., viscoelastography, has been used to estimate the shear modulus and viscosity of tissues by fitting the shear wave dispersion curve with a viscoelastic model, such as the Kelvin-Voigt model. Viscoelastography has been investigated for the assessment of liver fibrosis, breast lesions, and thyroid nodules (), and is an active area of research.
Polyvinyl alcohol (PVA) is widely used as a base material for ultrasound phantoms. A stable PVA hydrogel is formed through physical cross-linking during repeated freezing and thawing, producing a durable matrix whose acoustic and mechanical properties can be finely adjusted by varying additives and the number of freeze-thaw cycles (; ; ; ; ).
Aluminum oxide has been added to PVA phantoms as a scattering and attenuating agent (; ; ). The aluminum oxide concentration can be varied to tune the attenuation coefficient of PVA phantoms toward those of target tissues (; ). However, the implicit assumption is that aluminum oxide affects only attenuation, and its potential effect on other properties of PVA phantoms, such as the longitudinal sound speed and viscoelasticity, has not been systematically investigated.
Cryoprotectants, including ethylene glycol (EG) and propylene glycol (PG), have been added to modulate the physical cross-linking network of PVA phantoms. They alter the ice-crystallization process and suppress the formation of large ice crystals, giving an additional degree of control over microstructural properties (; ; ). Both EG and PG have been reported to modify the longitudinal sound speed (; ; ). recently showed that EG also alters the viscoelasticity of PVA phantoms. They found that incorporating a 20% (v/v) EG–based cryoprotectant in a PVA phantom produced a more homogeneous, less porous microstructure. The addition of the cryoprotectant significantly reduced both the shear modulus and viscosity relative to cryoprotectant-free phantoms, while also lowering the inter-sample variability of these estimates. By combining low- and high-viscosity PVA in the presence of the cryoprotectant, they further achieved a broad, tunable range of shear moduli at relatively constant viscosity. While PG has been used primarily to tune the longitudinal sound speed of PVA phantoms (), its concurrent effects on longitudinal wave attenuation and viscoelastic properties have not been evaluated (; ).
Although aluminum oxide and cryoprotectants are routinely incorporated into PVA phantoms, each additive has been used putatively to modulate a single property, i.e., either attenuation, sound speed, or microstructural homogeneity. Their potential concomitant effects on both the acoustic and viscoelastic properties of PVA phantoms have not been studied in depth. Accordingly, the goal of the present study is to systematically assess the effects of aluminum oxide and the two cryoprotectants, i.e., EG and PG, and their concentration dependence on the microstructural, acoustic, and viscoelastic properties of PVA phantoms. Phantoms were fabricated with either varying concentrations of aluminum oxide (0%–10% w/v) or varying concentrations of EG or PG (0%–20% v/v), with five independent samples per concentration group. The microstructure of each phantom was examined by scanning electron microscopy (SEM). The density, longitudinal sound speed, acoustic attenuation, shear modulus, and viscosity of the phantoms were then measured. The findings of this study contribute to the literature by offering new insights into the effects of these additives on the acoustic and viscoelastic properties of PVA phantoms. Overall, these efforts are consistent with one of the goals of the field, which is to improve measurement reproducibility by standardizing phantom fabrication methods and understanding confounding variables.
2 Materials and methods
2.1 Phantom fabrication
Polyvinyl alcohol phantoms were fabricated using previously published protocols (; ). A liquid base was first prepared by mixing distilled water with a cryoprotectant (EG or PG). The mixture was heated in a 700-W microwave for approximately 90 s until it reached 80 °C. PVA (10% (w/v), Elvanol 71–30, Kuraray America, Inc., Houston, TX, USA) aluminum oxide (<10 μm; Sigma 265497, USA), and silicon carbide (400 mesh; Sigma-Aldrich, USA) powders were then added slowly into the liquid mixture. Silicon carbide particles (2% (w/v)) served as acoustic scatterers. The mixture was stirred gently to disperse all powders without introducing air bubbles. To ensure complete dissolution of the PVA, the mixture was reheated in short 10-s bursts until it reached 90 °C, with close monitoring to prevent boiling over. It was then degassed in a vacuum chamber (approximately −1 bar) for at least 1 min to remove entrapped air, covered with aluminum foil, and allowed to cool to room temperature for ∼30 min. Any surface film that formed while cooling was removed.
The interior surfaces of the phantom molds were lightly greased with petroleum jelly to prevent leakage and facilitate the easy release of the phantoms. The molds were filled with the phantom mixture and covered with foil to limit water evaporation during the freeze-thaw cycle. The phantoms then underwent a single 24 h freeze-thaw cycle, consisting of pre-cooling at 4 °C for 30 min, freezing at −20 °C for 17.5 h, thawing at 4 °C for 4 h, and then at ambient temperature for the remaining 2 h. The resulting phantom was placed in a storage box and submerged in distilled water containing the same cryoprotectant concentration as the initial phantom mixture, to avoid the cryoprotectant leaching from the phantom. The phantoms were stored at room temperature for an additional 24 h prior to acoustic characterization.
Phantoms for density, longitudinal sound speed, and attenuation measurements had dimensions of 2.5 × 2.5 × 1.5 cm3. Larger samples were 7.0 × 4.3 × 2.5 cm3 to provide an adequate field of view for shear wave viscoelastography with a linear array transducer. A total of 75 phantoms were fabricated, divided equally into three studies with varying concentrations of (1) aluminum oxide, (2) EG, and (3) PG. Each study was comprised of five concentration groups, with five independent samples (n = 5) per group. Aluminum oxide concentrations were varied at 0, 2.5, 5, 7.5, and 10% (w/v). Both studies with cryoprotectants had varying concentrations of 0, 5, 10, 15, and 20% (v/v), which were within the range reported previously (; ).
2.2 Scanning electron microscopy (SEM)
Phantom morphology was examined by SEM. One-millimeter-thick samples were placed in plastic plates, covered with perforated film to allow aeration, frozen overnight at −80 °C, and lyophilized at −86 °C for 48 h to remove residual moisture by sublimation (; ). Dried samples were mounted on aluminum stubs with conductive adhesive and sputter-coated with a thin platinum layer. Surfaces were imaged on a field-emission microscope (JEOL JSM-7600F, Tokyo, Japan) at ×500 magnification (5 kV), acquiring five micrographs per specimen.
2.3 Density, longitudinal sound speed, and attenuation measurements
The density of each phantom was determined from its mass-to-volume ratio. Mass was measured with a precision analytical balance (SAB-203L, Scale-Tec, Vadodara, Gujarat, India), and volume was determined by the water-displacement method (; ).
Longitudinal sound speed and attenuation were measured with a broadband pulse-echo insertion-loss method in a degassed water bath (; ; ). An ultrasound impulse from a pulser-receiver (DPR300, JSR/Imaginant, Rochester, NY, USA) was transmitted and received with a single-element transducer (PA1415, 40-mm focal length, 5–20 MHz bandwidth, Precision Acoustics Ltd., Dorchester, United Kingdom). A stainless-steel block at the focus acted as an acoustic reflector (Figure 1).
FIGURE 1
A baseline reference echo was first recorded from the steel block in the absence of a phantom. A phantom sample was placed between the transducer and the reflector, and the attenuated echo was recorded. Signals were digitized on an oscilloscope (MDO3014, 100 MHz, 2.5 GS/s, Tektronix Inc.) and stored for analysis. The longitudinal sound speed was computed using Equation 1 ():where cw is the longitudinal sound speed in water at 22°C–24 °C (), Δt is the time shift introduced by the presence of the sample, and h is the sample thickness.
The attenuation spectrum was computed using Equation 2 ():where f is frequency and Vref and Vphantom are the echo amplitude spectra in the absence and presence of the phantom samples, respectively. The attenuation coefficient (dB/cm/MHz) was obtained as the slope of the linear fit to the attenuation spectrum over the 5–11 MHz band, which was the usable bandwidth of the linear array transducer used in subsequent shear wave viscoelastography studies. Both longitudinal sound speed and attenuation were measured at five independent locations of each independent phantom sample.
2.4 Shear wave viscoelastography measurements
The viscoelastic properties of the phantoms were estimated using shear wave viscoelastography, implemented on a programmable research ultrasound system (Vantage 128, Verasonics, Kirkland, WA, USA) (Figure 2). The protocol described by Bisht et al. was followed (). An acoustic radiation force impulse was generated with an L11-5v linear array using the center 32 active elements and a 1000-cycle push pulse at 5.2 MHz, focused at a 10 mm depth. The resulting shear waves were captured by single plane wave acquisitions (no angular compounding) at 7.6 MHz and 10,000 frames per second. Beamformed in-phase/quadrature (IQ) data from five independent imaging planes per phantom sample were saved. The post-processing workflow is presented in Figure 3. A region of interest (ROI) was selected from the reconstructed B-mode image, which was located at 8.2–11.8 mm axially (within the −6 dB depth of focus of the push beam) () and 0–10.1 mm laterally of the imaging plane. Axial particle displacements within the ROI were estimated using the two-dimensional Loupas autocorrelator (; ). spline interpolation was performed along the slow-time axis, and particle velocity was estimated by the finite difference method. A third-order Butterworth band-pass filter (10–800 Hz) and a median filter (kernel size 3λ) were then applied to reduce jitter and noise. A two-dimensional fast Fourier transform (2D FFT) was performed on the spatiotemporal particle velocity data. Shear wave phase velocity dispersion curves were generated using a routine adapted from the Radiological Society of North America Quantitative Imaging Biomarkers Alliance ultrasound shear-wave-speed repository (). The dispersion curve was analyzed over a frequency band, wherein the high-frequency cut-off was placed where the amplitude of the 2D FFT fell to 30% of its maximum, removing data with low signal-to-noise ratio. The low-frequency cut-off was placed where the difference between consecutive phase velocity values fell below 0.1 m/s, removing near-zero-frequency background noise (; ). The frequency range was then allowed to change dynamically until the coefficient of determination R2 was ≥0.85 when fitting the Kelvin-Voigt (KV) model to the shear wave dispersion curve (). The KV model was then used to estimate the shear modulus and viscosity of the phantoms. The KV model was selected because it is commonly used to model the viscoelastic properties of soft tissues (; ; ). The shear modulus, G, and the viscosity, η, were estimated using Equation 3 (; ):where ρ is the density and is the shear wave phase velocity as a function of the shear wave frequency (i.e., dispersion curve). A Levenberg–Marquardt scheme was used to fit the KV model to the dispersion curve. The shear modulus and viscosity were estimated from five independent imaging planes per phantom sample. All data analyses were performed in MATLAB (R2022a, The MathWorks, Inc., Natick, MA, United States) on a Windows operating system (AMD EPYC 7232P 8-Core Processor, 3.10 GHz, 32 GB RAM).
FIGURE 2
FIGURE 3
2.5 Statistical analysis
Statistical analyses were performed in MATLAB (R2022a) and GraphPad Prism (9.5.1, GraphPad Software, Boston, MA, USA). Density, longitudinal sound speed, attenuation coefficient, shear modulus, and viscosity were reported as the mean values of the parameter measurements across five imaging planes of five independent samples per group. The frequency dependence of the attenuation was characterized by linear regression, and the coefficient of determination R2 was used to assess the fit quality. Normality was verified using the Anderson-Darling test before group comparisons. One-way analysis of variance (ANOVA) was used to determine whether at least one group differed significantly across phantom groups. Subsequently, multiple comparisons were performed using Tukey’s honest significant difference test. Pearson correlation was used to determine whether a linear relationship existed between the additive concentration and the measured ultrasound parameters. A p-value of less than 0.05 was considered significant.
3 Results
3.1 Fabricated phantoms
Representative phantoms are shown in Figure 4. Samples differed in macroscopic appearance according to the content of aluminum oxide. The phantoms without aluminum oxide were gray (Figures 4A,B) due to the presence of silicon carbide particles. The phantoms with aluminum oxide were opaque and white (Figure 4C).
FIGURE 4
3.2 Phantoms with varying aluminum oxide concentration
3.2.1 Microstructure
Representative micrographs of phantoms with different aluminum oxide concentrations are shown in Figure 5. The samples without aluminum oxide had a comparatively smooth surface. Surface roughness increased progressively with increasing aluminum oxide concentration.
FIGURE 5
3.2.2 Density, longitudinal sound speed, and attenuation measurements
Density measurements are presented in Figure 6A, with sample means ranging from 1.03–1.14 g/cm3. The data within each group were found to approximate a normal distribution. The ANOVA and Tukey’s post hoc test results indicated a significant effect of aluminum oxide content on density. Pearson correlation showed a strong positive relationship between aluminum oxide concentration and density (r = 0.91, p < 0.001). Longitudinal sound speeds ranged from 1576–1583 m/s (Figure 6B). The differences in sound speed measurements between groups of various aluminum oxide concentrations were not significant (p = 0.082). Attenuation increased linearly with frequency over the 5–11 MHz band (Figure 6C). The attenuation coefficient measurements ranged from 0.12 to 0.54 dB/cm/MHz across aluminum oxide concentrations (Figure 6D), which were significantly different across all concentration groups. A Pearson correlation coefficient of 0.98 indicated a strong positive linear relationship (p < 0.001) between the attenuation coefficient and aluminum oxide concentration.
FIGURE 6
3.2.3 Viscoelasticity measurements
The shear wave viscoelastography measurements across aluminum oxide concentrations are shown in Figure 7. Shear modulus estimates across samples ranged between 3.05 and 5.32 kPa (Figure 7A) and had a significant positive correlation with aluminum oxide concentration (r = 0.79, p < 0.001). Viscosity measurements ranged from 1.58 to 2.15 Pa s (Figure 7B). However, the correlation between viscosity and aluminum oxide concentration was not significant (r = 0.32, p = 0.12).
FIGURE 7
3.3 Phantoms with varying cryoprotectant concentrations
3.3.1 Scanning electron microscopy images
Scanning electron micrographs of phantoms prepared with EG and PG are shown in Figure 8. The control samples (i.e., 0% cryoprotectant) presented a highly porous hydrogel network characteristic of a PVA gel matrix. On the other hand, the addition of EG produced a distinct shift to a dense, non-porous structure that persisted across all tested concentrations. Specific microstructural differences between EG concentrations could not be resolved by SEM. Phantoms showed the same porous-to-non-porous transition upon addition of PG (Figure 8B), wherein the non-porous structure maintained across the full concentration range. The PVA matrix appeared to reach morphological saturation even at low cryoprotectant concentrations such that differences in microstructure between concentrations were not visually discernible.
FIGURE 8
3.3.2 Density, longitudinal sound speed, and attenuation measurements
Density measurements of the phantoms with different concentrations of the cryoprotectants are shown in Figure 9A. Phantoms with EG had densities spanning 1.05–1.07 g/cm3, and those with PG ranged from 1.066 to 1.074 g/cm3. All data approximated normal distributions (p > 0.05), and one-way ANOVA indicated that the differences in density measurements across groups with various cryoprotectant concentrations were not significant (p = 0.58 for EG and p = 0.80 for PG).
FIGURE 9
Longitudinal sound speed measurements are shown in Figure 9B and ranged from 1535 to 1643 m/s for phantoms with EG and from 1535 to 1666 m/s for those with PG. All groups were normally distributed (p > 0.05). Longitudinal sound speed measurements differed significantly across all concentration pairs (p < 0.05). Correlation analysis revealed a strong positive linear relationship between the sound speed measurements and cryoprotectant concentration for both EG (r = 0.99, p < 0.001) and PG (r = 0.99, p < 0.001). Attenuation coefficients for phantoms with 0%–20% (v/v) EG and PG are shown in Figure 9C, with sample means ranging from 0.27–0.40 dB/cm/MHz and from 0.27–0.39 dB/cm/MHz, respectively. The Anderson-Darling test confirmed normality, and one-way ANOVA showed significant differences across concentrations for both cryoprotectants (p < 0.0001). Tukey’s post hoc comparisons identified significant differences between the pairs listed in Tables 1, 2. Correlation analysis indicated a significant positive relationship between attenuation and cryoprotectant concentration for both EG (r = 0.84, p < 0.001) and PG (r = 0.82, p < 0.0001).
TABLE 1
| Concentration of EG (% v/v) | p-value |
|---|---|
| 0 and 10 | 0.0390 |
| 0 and 15 | 0.0121 |
| 0 and 20 | <0.0001 |
| 5 and 15 | 0.0477 |
| 5 and 20 | <0.0001 |
| 10 and 20 | 0.0019 |
| 15 and 20 | 0.0064 |
Statistical significance (p-values) for pairwise comparisons of attenuation between EG concentrations.
TABLE 2
| Concentration of PG (% v/v) | p-value |
|---|---|
| 0 and 15 | 0.0085 |
| 0 and 20 | <0.0001 |
| 5 and 15 | 0.0303 |
| 5 and 20 | 0.0002 |
| 10 and 20 | 0.0022 |
Statistical significance (p-values) for pairwise comparisons of attenuation between PG concentrations.
3.3.3 Viscoelasticity measurements
Estimated shear moduli over the 0%–20% range for both cryoprotectants are shown in Figure 10A. Note that the 0% EG and 0% PG groups are the same phantom samples, as they do not contain any cryoprotectant. All datasets passed the normality test (p > 0.05). For PG, compared to 0% (v/v), the shear modulus was significantly lower at 10% (p < 0.0001), 15% (p < 0.0001), and 20% (p < 0.0001). Significant differences were also observed between the 5% group and all higher concentrations (p < 0.0001). However, the differences between the shear modulus values between 10, 15, and 20% were not significant (p > 0.05). EG produced a non-monotonic response, wherein the shear modulus decreased progressively from 0% to 15%, reaching a minimum sample mean of 3.81 kPa, and then increased sharply to 8.01 kPa at 20%. The 20% EG group differed significantly from all other phantom types (p < 0.0001).
FIGURE 10
The viscosity measurements are shown in Figure 10B. Values ranged from 0.93 to 1.69 Pa s for phantoms with EG and 0.90–1.69 Pa s for those with PG. All datasets were found to approximate normal distributions (p > 0.05). For E.G., adding cryoprotectant decreased viscosity significantly relative to the 0% group (c), but no significant differences were found between the 5, 10, 15, and 20% groups, indicating that phantom viscosity was altered by the initial introduction of EG and then remained stable between 5% to 20%. For PG, significant differences in viscosity estimates emerged only between 0% and 5% (p < 0.0001), 0% and 10% (p < 0.0001), 0% and 15% (p < 0.0001), 0% and 20% (p < 0.05) groups, with no significant differences between the groups with PG. Both cryoprotectants modulated viscosity similarly, with values that were comparable across all tested concentrations.
4 Discussion
Well-characterized phantom development approaches are essential for reliable assessment and benchmarking of ultrasound elastography and viscoelastography approaches. This study was conducted to determine how aluminum oxide and two cryoprotectants, EG and PG, affected the acoustic and viscoelastic properties of PVA tissue-mimicking phantoms. Pure PVA hydrogels provide a baseline matrix, but their acoustic and mechanical properties do not consistently fall within the soft-tissue range (). In the present work, adding aluminum oxide and cryoprotectants to PVA phantoms modulated the sound speed, attenuation, and viscoelastic properties. Notably, we report an increase in shear modulus as aluminum oxide concentration increased and demonstrated the effects of cryoprotectants on both attenuation and viscoelastic properties, which have not been explored previously.
4.1 Effects of aluminum oxide
As shown in SEM images, phantoms without aluminum oxide had relatively smooth hydrogel surfaces (Figure 5). Increasing aluminum oxide content produced progressively more irregular surfaces, which were consistent with previously reported roughening of PVA phantoms loaded with rigid scatterers (; ). The rigid particles embedded in the soft matrix can disrupt the continuous polymer network and produce the observed roughness, which could be a physical basis for the concentration-dependent acoustic and mechanical behavior. Density increased with aluminum oxide concentration and remained within the soft-tissue range (Figure 6A), comparable to reported PVA phantoms, because the solid filler raised the composite mass-to-volume ratio (; ).
Attenuation increased linearly with frequency over 5–11 MHz (Figure 6C), and the attenuation coefficient spanned within the soft-tissue interval (; ). The very strong correlation (r = 0.98) and the significant differences across every concentration pair confirm that aluminum oxide is an effective agent for tuning attenuation across a tissue-relevant range. In contrast, the longitudinal sound speed was largely insensitive to aluminum oxide (Figure 6B) and remained within the soft-tissue range, in agreement with reported PVA-phantom values (; ). This insensitivity is useful for phantom design because attenuation can be adjusted through aluminum oxide without shifting the sound speed out of the physiological range.
On the other hand, the shear modulus increased with aluminum oxide concentration (Figure 7A), a notable finding in the study. This increase may be due to the rigid aluminum oxide particles, which do not deform under shear and restrict the conformational mobility of the surrounding polymer chains, thereby progressively constraining the network and increasing the force required for deformation (). Although aluminum oxide is usually added solely to modify attenuation, these results show that it also leads to a concomitant increase in the elasticity of the PVA phantoms. Hence, this effect should be considered during phantom design and characterization of elastography approaches to avoid potential measurement bias.
Meanwhile, viscosity did not change significantly with aluminum oxide content (Figure 7B) and remained within the soft-tissue range (), consistent with reported PVA phantoms (; ). Taken together, increasing aluminum oxide raises attenuation, density, and shear modulus, while leaving sound speed and viscosity essentially unchanged. These findings show that aluminum oxide cannot be treated purely as an attenuation agent.
4.2 Effects of cryoprotectants
SEM images indicated that phantoms without cryoprotectant had a highly porous network, whereas phantoms containing EG or PG were non-porous across all tested concentrations (Figure 8). These findings are in agreement with prior reports of smoother, more homogeneous microstructures for EG-containing PVA phantoms (; ). Cryoprotectants lower the freezing temperature of the precursor solution and limit the growth of large ice crystals that would otherwise create porous inhomogeneities in the cryogel. Because all cryoprotectant phantoms were uniformly non-porous, SEM alone could not resolve concentration-specific microstructural differences. Density remained within the soft-tissue range (Figure 9A), comparable to reported PVA phantoms with cryoprotectants (; ).
Sound speed rose markedly with cryoprotectant concentration (Figure 9B), consistent with reports of higher longitudinal sound speeds when glycols are added to PVA (; ). The higher intrinsic sound speed of glycols relative to water and the increased bulk modulus of the interstitial fluid plausibly account for the effect, as glycol hydroxyl groups form hydrogen bonds with water and with the PVA chains. PG reached a slightly higher maximum sound speed (1666 m/s) than EG (1643 m/s), possibly because its additional methyl group produces a tighter local arrangement within the hydrogel pores (; ).
Attenuation increased with cryoprotectant concentration for both cryoprotectants (Figure 9C). Although cryoprotectants have been incorporated primarily to suppress ice-crystal formation and to control sound speed (; ), the present results show that EG and PG also raise attenuation in a concentration-dependent manner. These results likely reflect the denser hydrogen-bonded fluid network formed as the glycol fraction rises, which increases the absorptive component of attenuation as propagating waves lose more energy to friction within the more tightly bonded fluid phase (; ). In practice, cryoprotectant concentration cannot be selected based on microstructure or sound speed alone, as it also affects attenuation.
The shear modulus showed a distinctly non-monotonic dependence on cryoprotectant concentration. For EG, it decreased from 6.94 kPa at 0% to a minimum of 3.81 kPa at 15% before rising sharply to 8.01 kPa at 20%, whereas for PG, it decreased from 6.94 kPa at 0% and saturated to around 4 kPa from 10% to 20% (Figure 10A). The initial softening in both glycols is consistent with a plasticization mechanism, in which glycol molecules insert between PVA chains, disrupt interchain hydrogen bonds, increase free volume, and produce a softer, more compliant network (; ). The sudden stiffening of EG phantoms at 20% may reflect a concentration threshold at which the small EG molecule induces a localized dehydration effect, drawing water away from the PVA chains. In the absence of sufficient interstitial water to separate them, the chains may re-establish strong intermolecular cross-links or form microcrystallites, producing the observed stiffening. The bulkier PG molecule (molar mass 76.09 g/mol versus 62.07 g/mol for EG) introduces greater steric hindrance and less efficient chain packing, which may likely explain why this re-crystallization is not observed for PG within the tested range (). This behavior is directly relevant to the design of elastography phantoms. The choice between EG and PG, as well as the specific cryoprotectant concentration, strongly determines the viscoelastic properties. In particular, EG exhibited a non-monotonic response that must be anticipated when working toward 20% (v/v).
Viscosity decreased significantly from the 0% baseline upon addition of either cryoprotectant and then plateaued at higher concentrations (Figure 10B), remaining within the soft-tissue range and comparable to reported PVA phantoms (; ; ). Specifically, for E.G., the 0% group differed from all other groups (p < 0.05), with no significant differences between 5, 10, 15, and 20% E.G., For PG, significant differences were observed only between the 0%, 5%, and 10% groups. The plateau is consistent with the initial addition of glycol-structured free water within the hydrogel pores through hydrogen bonding (; ), thereby increasing resistance to shear flow (). Once the primary hydration and hydrogen-bonding sites are saturated (around 5%–10% v/v), glycol pools into the already-structured fluid phase without substantially changing the macroscopic viscosity (; ). As with shear modulus, the cryoprotectant concentration must therefore be selected with awareness of its coupled effect on viscoelastic properties rather than based on its ice-crystal-inhibiting role alone.
4.3 Contributions of the work
The ultrasonic and shear-wave elastographic characterization of viscoelastic PVA phantoms and the role of curing and cryoprotectants have been reported in the literature. Previous work by has also assessed the effects of the absence and presence of EG (i.e., 0% and 20% v/v) on the acoustic and shear-wave viscoelastography measurements in PVA phantoms (). However, they did not systematically investigate and quantify the effect of a range of EG concentrations. The present study has reported on this aspect and evaluated another cryoprotectant, i.e., PG, as well as aluminum oxide, which is typically used to modulate acoustic attenuation in PVA phantoms.
Prior work has also modified the structural properties of PVA phantoms. fabricated and characterized the mechanical properties of an anisotropic PVA phantom by stretching the PVA polymer network during freeze-thaw cycling (). Subsequently, developed anisotropic viscoelastic PVA phantoms by embedding dialysis fibers and quantified the effects of fiber angle on shear wave viscoelastography measurements (). While these prior studies focused on the effects of anisotropy in PVA phantoms, the current work examines a different aspect, i.e., the effects of the additives, aluminum oxide and two cryoprotectants (EG and PG), on the acoustic and mechanical properties of these phantoms.
The novelty of this work lies in the systematic characterization of the (1) microstructural (via scanning electron microscopy), (2) ultrasonic (via longitudinal sound speed and acoustic attenuation measurements), and (3) viscoelastic properties (via shear wave viscoelastography) of PVA phantoms containing aluminum oxide and EG or PG. Specifically, this work contributes to the literature on PVA phantoms as follows: First, the effect of aluminum oxide on the ultrasonic and viscoelastic properties of PVA phantoms was identified. In the range of 0%–10% (w/v) aluminum oxide concentrations, not only did the acoustic attenuation change, but also the shear modulus of the PVA phantoms increased by 1.7-fold (3.05–5.32 kPa; r = 0.79, p < 0.001). Hence, we demonstrate that aluminum oxide significantly affects the mechanical properties of PVA phantoms, a finding not reported previously in the literature. These results are significant because attenuation and mechanical properties cannot be independently tuned using aluminum oxide. These results have a direct impact on efforts to avoid confounding effects in elastography quality-control phantom studies, in which PVA phantoms doped with aluminum oxide to match tissue attenuation can simultaneously exhibit changes in mechanical properties.
Secondly, we demonstrated the effect of PG on the ultrasonic and viscoelastic properties of PVA phantoms. PG has been used in PVA phantoms solely to tune the longitudinal sound speed (). In the present work, we show that PG also increases acoustic attenuation and alters both shear modulus and viscosity of PVA phantoms. Further, we report the first direct comparison of EG and PG under identical fabrication and measurement conditions. Third, we report a unique non-monotonic relationship between EG concentration and the shear modulus of PVA phantoms. Specifically, shear modulus measurements decreased with EG concentration to a minimum of 3.81 kPa at 15% v/v before rising sharply to 8.01 kPa at 20% v/v. However, the shear modulus measurements of phantoms with PG did not exhibit such a reversal within the same range of PG concentrations. Taken together, our findings add new insights to the literature on PVA phantoms, which can help clarify confounding variables, improve reproducibility, and assist in standardizing PVA phantom fabrication methods.
4.4 Limitations and future directions
Only the cryoprotectant concentrations previously reported for PVA phantoms (0%–20% (v/v)) were investigated in this work. Subsequent studies can also examine EG at concentrations above 20% to determine whether the shear modulus continues to rise or eventually reverses. Alternative cryoprotectants and sugar alcohols that suppress ice-crystal formation and tune sound speed while minimally affecting the mechanical properties of the phantoms can also be evaluated. This study characterized the acoustic and viscoelastic properties of the PVA phantoms within 2 days of fabrication. Therefore, the long-term stability of the phantoms was not assessed. Longitudinal studies over 6–12 months can be conducted to monitor the acoustic and mechanical stability of these phantoms.
Various rheological models have been reported for estimating the viscoelastic properties of phantoms and tissues (). In the present study, the Kelvin-Voigt model was used because it has been widely reported in the literature. However, other rheological models may better represent the dispersive behavior of our PVA phantoms. As the primary focus of the present work is to systematically identify the effects of aluminum oxide and two cryoprotectants on the ultrasonic and viscoelastic estimates of PVA phantoms, other rheological models were not explored in the current study. Further investigations should be performed with other rheological models suitable for viscoelastic PVA phantoms.
Another limitation of the current work was that shear wave attenuation was not accounted for, which could potentially bias the viscoelastic estimates of the PVA phantoms (; ). Regarding the wave geometry, the ROI was confined within the −6 dB depth of focus of the ARFI push beam to approximate a planar shear wave propagation. Thus, the effect of diffraction and geometric spreading can be reduced. Further, the ROI, push parameters, and focal depth were held constant across all phantom measurements to minimize confounding effects between samples. Future work will elucidate the inherent coupling between dispersion, attenuation, and geometric effects and account for shear wave attenuation using approaches, such as the Cole-Cole framework () and the Kramers–Kronig relationships (), which can overcome the inherent constraints of dispersion curve fitting.
Previous work by reported a comparison of rheometry and shear-wave viscoelastography estimates of PVA phantoms that have undergone one to three freeze-thaw cycles (). They reported that shear modulus estimates from shear wave viscoelastography were not significantly different from those obtained by rheometry. However, the viscosity estimates differed by 25%–50% between these two methods. This discrepancy was likely due to differences in operating frequency ranges between rheometry (1–16 Hz) and shear wave viscoelastography (100–700 Hz). Prior work by also reported differences between rheometry and shear wave elastography measurements, which they also attributed to differences in frequency ranges. Although the PVA phantom composition in the present study differed from that of , the phantom fabrication protocol and shear wave viscoelastography algorithm were the same. Hence, independent rheometry measurements were not performed in the present work due to potential differences in absolute values between rheometry and shear-wave viscoelastography measurements.
5 Conclusion
This study demonstrates that the acoustic, mechanical, and microstructural properties of polyvinyl alcohol (PVA) phantoms are modified with the addition of aluminum oxide and cryoprotectants, EG and PG. While aluminum oxide is traditionally used to tune acoustic attenuation, this work shows that it also affects viscoelastic properties. Furthermore, although EG and PG have been known to prevent ice-crystal formation and increase sound speed, they also introduce non-monotonic alterations to attenuation and viscoelasticity. These findings inform researchers to design and tune multi-parameter phantoms for various ultrasound imaging and elastography applications accurately.
Statements
Data availability statement
The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.
Author contributions
DG: Formal Analysis, Writing – original draft, Software, Data curation, Investigation, Writing – review and editing, Methodology, Conceptualization, Visualization, Validation. AC: Writing – review and editing, Investigation, Writing – original draft, Software, Formal Analysis, Visualization, Data curation, Methodology. JP: Writing – review and editing, Software, Writing – original draft, Formal Analysis, Visualization, Data curation, Methodology, Validation, Investigation. KM-S: Conceptualization, Funding acquisition, Investigation, Writing – review and editing, Writing – original draft, Supervision, Software, Data curation, Project administration, Validation, Resources, Formal Analysis, Methodology, Visualization.
Funding
The author(s) declared that financial support was received for this work and/or its publication. This work was supported by the India Alliance, Department of Biotechnology (Grant No. IA/TSG/23/1/600493).
Acknowledgments
The authors thank Prof. Himanshu Shekhar for helpful discussions, and the Central Instrumentation Facility (CIF) at the Indian Institute of Technology Gandhinagar for support.
Conflict of interest
The author(s) declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Generative AI statement
The author(s) declared that generative AI was not used in the creation of this manuscript.
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Keywords
tissue-mimicking phantom, polyvinyl alcohol, aluminum oxide, cyroprotectant, ethylene glycol, propylene glycol, acoustic attenuation, shear wave elastography
Citation
Gupta D, Chandra A, Patil JM and Mercado-Shekhar KP (2026) Characterization of tissue-mimicking polyvinyl alcohol phantoms for shear wave viscoelastography: effects of aluminum oxide and cryoprotectant concentration. Front. Acoust. 4:1920457. doi: 10.3389/facou.2026.1920457
Received
26 June 2026
Revised
31 August 2026
Accepted
03 September 2026
Published
29 September 2026
Volume
4 - 2026
Edited by
Chengzhi Shi, University of Michigan, United States
Reviewed by
Ramon Risco, Spanish National Research Council (CSIC), Spain
Siladitya Khan, University of Rochester, United States
Updates
Copyright
© 2026 Gupta, Chandra, Patil and Mercado-Shekhar.
This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Karla P. Mercado-Shekhar, [email protected]
Disclaimer
All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher.
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