Anisotropic Polyvinyl Alcohol Phantom Fabrication for Ultrasound Elastography: Procedure and Quality Controls

Summary

This protocol describes a robust, reproducible method for fabricating anisotropic polyvinyl alcohol (PVA) phantoms—essential tools for validating ultrasound elastography techniques targeting mechanically anisotropic soft tissues (e.g., skeletal muscle).

Abstract

Polyvinyl alcohol (PVA) phantoms are typically isotropic and are widely used to validate ultrasound elastography techniques for soft-tissue evaluation. However, some biological soft tissues (e.g., skeletal muscle) exhibit distinct mechanical anisotropy, which necessitates the use of anisotropic PVA phantoms for the rigorous testing of elastography methods targeting these tissues. While prior studies have noted that in-house anisotropic PVA phantoms can be fabricated via stretch-integrated freeze/thaw cycles (FTCs), critical technical details (e.g., fabrication process, quality control) remain insufficiently documented. This work presents a visually detailed, reproducible protocol for fabricating anisotropic PVA phantoms, focusing on key materials, stepwise processes, and quality controls to induce stable, uniform anisotropy. Key materials include PVA as the phantom matrix, potassium sorbate as a preservative, graphite particles as acoustic scatterers, and pure water (or deionized water) as the solvent. The fabrication process comprises three core stages: 1) Preparation of a homogeneous PVA-based solution through controlled thermal conditions to ensure complete PVA dissolution; 2) Solidification via FTCs: the cooled solution is poured into 3D-printed molds, followed by stretch-free FTCs to form a preliminary structure; 3) Inducing anisotropy via stretched FTCs: additional FTCs are performed under controlled stretching to induce directional anisotropy. Quality-control measures (e.g., avoiding air bubbles during PVA dissolution) are described in detail. After fabrication, ultrasound shear wave imaging (SWI) and uniaxial tensile testing are employed to confirm the phantom's mechanical anisotropy. This paper provides a standardized approach for fabricating anisotropic tissue-mimicking phantoms to validate ultrasound elastography techniques with enhanced accuracy and consistency.

Introduction

Certain biological soft tissues (e.g., skeletal muscle) exhibit macroscopic mechanical anisotropy, which originates from their aligned microstructural architecture¹. Specifically, connective tissues organize muscle fibers into a hierarchical structure: endomysium surrounds individual fibers, perimysium bundles fibers into fascicles, and epimysium encapsulates the entire muscle, supporting force transmission and nutrient supply². Ultrasound-based elastography techniques, such as shear wave imaging (SWI), are widely used for muscle assessment^3,4. However, the anisotropic mechanical properties of muscle theoretically result in direction-dependent shear wave propagation⁵, which complicates the quantification of muscle stiffness⁶. Mechanically anisotropic tissue-mimicking phantoms are essential for advancing ultrasound elastography in muscle assessment.

Polyvinyl alcohol (PVA) is an ideal material for such phantoms, as maintaining mechanical stretch during freeze/thaw cycles (FTCs) aligns PVA molecular chains, thereby inducing mechanical anisotropy^7,8. Conventional approaches that use simple PVA cuboids under stretching often lead to inhomogeneous strain due to surface slippage, thereby compromising reproducibility. This limitation can be addressed by employing an optimized phantom geometry with end holding rings, which ensures reliable stretching and uniform strain distribution⁹—markedly enhancing the quality of anisotropic PVA phantoms. In this study, we provide a detailed visual demonstration of the fabrication of such anisotropic PVA phantoms and validate their mechanical anisotropy via both SWI and uniaxial tensile testing.

Protocol

NOTE: The following steps are illustrated in Figure 1.

1. 3D printing of phantom mold

1. Using 3D modeling software, design the phantom geometry to incorporate two holding rings (one at each end) for maintaining stretch, with an effective central region of 35 mm × 60 mm × 5 mm (length × width × height) to ensure uniform anisotropy induction.
2. Print the mold using a 3D printer.
   NOTE: The holding rings prevent slippage during stretching, ensuring consistent strain distribution. All 3D printing files (.stl) are provided in Supplemental File 1.

2. Preparation of PVA solution

1. Weigh PVA powder (10 wt%), potassium sorbate (1 wt%), and graphite particles (1 wt%, ~6.5 µm diameter), and measure the corresponding volume of pure water (deionized water is recommended) to prepare a batch volume of ~200 mL.
2. Combine PVA powder and pure water in a beaker, place the beaker on a magnetic stirrer equipped with a stir bar, cover the beaker with aluminum foil, and stir at ~600 rpm at room temperature for 10 min until PVA is fully dispersed.
3. Transfer the beaker to an 80 °C water bath and continue heating until PVA is completely dissolved (evidenced by a clear, particle-free solution).
   NOTE: Wear heat-resistant gloves and eye protection gear during heating to prevent thermal burns. Ensure no undissolved PVA particles remain, as they will compromise phantom homogeneity.

3. Addition of additives and casting

1. Cool the solution to ~40 °C while continuing to stir at ~600 rpm on the magnetic stirrer.
2. Remove the aluminum foil, add potassium sorbate and graphite particles, re-cover with foil, and stir at ~600 rpm until the additives are uniformly dispersed (visually confirm homogeneous distribution).
3. (Optional) Degas the PVA solution to remove residual air bubbles. Place the beaker containing the mixed solution in a vacuum chamber and apply a vacuum for approximately 30 min to remove air bubbles from the PVA solution.
4. Cast the solution into the 3D-printed mold and assemble the mold using screws.
   NOTE: Avoid introducing air bubbles during casting. Gently tap the mold if necessary to eliminate trapped air bubbles inside the solution.

4. Initial FTC for PVA solidification

1. Place the mold in a −20 °C freezer for 12 h.
2. Remove the mold from the freezer and thaw at room temperature (20 °C) for 12 h.
3. Carefully demold the phantom to prevent damage.
   NOTE: This step forms an isotropic base phantom, providing a stable foundation for subsequent anisotropy induction.

5. Inducing anisotropy via stretched FTCs

1. Place the phantom on the custom-made stretching device, gradually stretch it to 180% of its effective length (80% tensile strain), measure the stretching distance manually, and lock the device to prevent retraction.
2. Transfer the entire stretching device (with the phantom secured) into a −20 °C freezer for 12 h, followed by thawing at room temperature (20 °C) for 12 h.
3. Repeat this FTC cycle once more under sustained stretching.
   NOTE: The custom-made stretching device is made of a commercially available bidirectional, opposite-thread linear stage assembled with two 3D-printed phantom-holding mounts, for which the STL files are provided in Supplemental File 1. Wear insulated cold-protective gloves to avoid frostbite when removing the stretching device from the −20 °C freezer. Sustained stretching during FTCs aligns PVA molecular chains unidirectionally. Total FTCs employed in this study: one stretch-free cycle + two stretched cycles (80% tensile strain).

6. Final release and storage

1. Following the final FTC thawing step, gently release the stretching device to prevent recoil-induced damage.
2. Remove the phantom from the stretching device. The anisotropic PVA phantom is now complete.
3. Store the phantom immersed in water inside a sealed plastic container at 4 °C.

Results

Figure 2A presents a schematic of the complete fabrication process, including PVA solution preparation, molding, initial stretch-free FTC, demolding, and subsequent stretched FTCs. Using the proposed protocol, we fabricated two anisotropic PVA phantoms for SWI and uniaxial tensile testing, respectively. SWI was performed using a research ultrasound system equipped with an L7-4 probe (center frequency: 5.2 MHz), employing supersonic mode (3 foci, 100 µs push)⁴ and cohe...

Discussion

The success of this protocol relies on achieving complete, uniform PVA dissolution and maintaining sustained stretching during FTCs—two critical steps for inducing stable, homogeneous mechanical anisotropy. The recipe used herein (i.e., PVA concentration, FTC number, and applied tensile strain) was selected to fabricate anisotropic PVA phantoms with stiffness within the physiological range of muscle tissue. Notably, the stiffness and anisotropy ratio of the PVA phantom can be readily tuned by adjusting these recipe param...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Analytical balance	OHAUS	AX224	Analytical balance with internal calibration, capacity 220 g, readability 0.1 mg
Bidirectional, opposite-thread linear stage	Taobao	N/A	Travel range 250 mm for each thread, used for constructing the custom-made stretching device
FDM 3D Printer	Bambu Lab	X1 Carbon	Used for printing the phantom mold, as well as the phantom?holding mounts for the custom-made stretching device
FreeCAD	FreeCAD Open-Source Community	N/A	Version 0.22.0, used for designing the phantom mold and the phantom-holding mounts of the custom-made stretching device
Graphite particles	MREDA	M151649	Graphite particles, 99.95% purity, 2000 mesh(~6.5 um), used as acoustic scatterers, 1% by weight
Magnetic stir bar	Corning	401435	PTFE-coated magnetic stir bar, 1 x 3/8 inch, for stirring PVA solution
Magnetic stirrer	IKA	25005927	RCT basic safety control magnetic stirrer with heating function
Polyvinyl alcohol	MACKLIN	P81584	Polyvinyl alcohol, Mw 89000-98000, 95% hydrolyzed, used as phantom matrix, 10% by weight
Potassium sorbate	Sigma-Aldrich	85520	Potassium sorbate,≥99.0% purity, used as preservative, 1% by weight
Rotation stage	Newport	URS50BPP	Used for rotating the ultrasound probe in shear wave imaging test for multipe probe angles (0°:2°:90°)
Single column universal testing machine	Instron	Model 5944	Used for uniaxial tensile testing, 50 N force sensor
Research Ultrasound System	Verasonics	Vantage 256	Used for conducting shear wave imaging test, L7-4 probe (center frequency: 5.2 MHz)
Water bath	LICHEN	HH-2	Digital display constant temperature water bath, 2 holes single row, for heating and dissolving PVA solution