3D printable anthropomorphic phantom for mimicking the dielectric properties of mammary tissues and use thereof

Abstract

The present invention relates to a breast phantom, comprising: a first hydrogel that forms a simulated phantom of normal breast tissue and a second hydrogel that forms a simulated phantom of cancerous breast tissue, wherein the first hydrogel comprises cross-linked 2-hydroxyethyl acrylate and the second hydrogel comprises cross-linked methacrylate hyaluronic acid. The present invention also relates to the method of preparing said phantom and to its use.

Description

[0001] DESCRIPTION

[0002] A 3D Printable Anthropomorphic Phantom to Mimic the Dielectric Properties of Breast Tissue and its Use

[0003] The invention relates to an anthropomorphic phantom that is manufactured by 3D printing and assembly of tissue-simulating materials that mimic the dielectric properties of common breast tissue, making it particularly suitable for use in microwave imaging (MI).

[0004] Therefore, the present invention belongs to the field of medicine.

[0005] STATE OF THE ART

[0006] Breast cancer (BC) remains a major global health problem, affecting millions of women and causing considerable morbidity and mortality. Early detection of breast cancer is crucial for improving patient outcomes, as it allows for timely interventions and personalized treatment strategies. Traditional imaging modalities, such as 2D X-ray mammography, and recent advances like 3D digital breast tomosynthesis have played a crucial role in breast cancer screening and diagnosis. These techniques are employed in population-based screening programs, as they have been shown to reduce BC mortality. However, these methods rely on ionizing X-rays, which limits their frequency (typically every two years) and age range (between 45 and 74 years), in addition to presenting several limitations, such as their reduced ability to differentiate between benign and malignant lesions in dense breasts.

[0007] Microwave imaging (MI) has emerged as a promising non-ionizing imaging modality for breast cancer detection, offering potential advantages over traditional techniques. It uses electromagnetic waves within the microwave frequency range to examine breast tissue and create images based on the tissues' dielectric properties. Unlike methods based on ionizing radiation, microwave imaging carries no risk of exposure to this type of radiation, making it safer for repeated examinations and a suitable option for young women and high-risk populations. Despite its potential, microwave imaging faces challenges that have hindered its widespread adoption in clinical practice. A significant limitation is the inherent complexity of microwave-tissue interactions, which generates artifacts in the images and reduces their quality.Furthermore, the dispersion and absorption properties of breast tissue vary considerably between individuals, which affects the accuracy and consistency of the results of this new technique.

[0008] To address the limitations of microwave imaging and facilitate its development and validation, the use of anthropomorphic phantoms has gained significant attention. Medical imaging phantoms are artificial constructs designed to replicate the properties and behavior of human tissues in various imaging modalities. These phantoms can be computational or physically constructed and serve as valuable tools for calibrating, validating, and optimizing conventional and emerging imaging technologies, as well as for conducting research and development in the field of medical imaging. Thus, imaging phantoms are crucial tools that bridge the gap between new imaging technologies and clinical practice by accurately and reproducibly emulating human tissues.They allow researchers and clinicians to explore the full potential of imaging techniques, leading to optimized diagnostic procedures that ultimately contribute to better patient outcomes and optimal healthcare practices.

[0009] In relation to the state of the art, phantoms play a crucial role in the field of radiology, serving as indispensable tools for a wide range of applications. These specialized objects, which mimic the properties of human tissues and structures, are used extensively in conventional imaging techniques to evaluate and improve the performance of X-ray, computed tomography (CT), and magnetic resonance imaging (MRI) systems. They help establish image quality, optimize radiation dose, and validate image reconstruction algorithms, among other applications.

[0010] In recent years, imaging phantoms have gained prominence in emerging modalities such as ultrasound, optical imaging, positron emission tomography (PET), single-photon emission computed tomography (SPECT), and microwave imaging. Phantomes designed for ultrasound imaging, for example, mimic tissue structures and acoustic properties, facilitating the evaluation of image resolution, penetration, and artifacts. Similarly, in molecular imaging, PET and SPECT phantoms allow researchers to validate radiopharmaceutical distribution and quantify tracer uptake in specific tissues. In optical imaging, phantoms replicate the light scattering and absorption properties of tissues, enabling researchers to fine-tune diffuse optical and fluorescence imaging systems.In the context of microwave imaging, phantoms mimic the dielectric properties of biological tissues, supporting the development and validation of breast cancer detection systems and other biomedical applications.

[0011] The prevailing trajectory in the advancement of phantoms focuses on the creation of high-performance computational phantoms (Kainz, W. et al. (2019) IEEE Transactions on Radiation and Plasma Medical Sciences, 3(1), 1-23), and the construction of physical prototypes using 3D printing technology (Filippou, V., & Tsoumpas, C. (2018). Medical Physics, 45(9)). Despite significant progress in this area, the search for refined and versatile phantom models, especially for emerging techniques such as microwave imaging, persists as researchers seek to address the challenges and complexities posed by these new technologies. The evolution of microwave imaging techniques requires the creation of phantoms that can simulate the diverse and intricate electromagnetic interactions that occur within the human body.These phantoms are valuable tools for validating the performance of systems and algorithms in this new imaging technique, ensuring their reliability and accuracy in real-world scenarios. The literature describes two main types of phantoms: (1) homogeneous phantoms made of a single material with uniform dielectric properties, which can be used to calibrate IM systems and evaluate their basic performance; and (2) heterogeneous phantoms made of multiple materials with different dielectric properties to mimic the complex structure of biological tissues. Furthermore, three types of materials are commonly used to fabricate phantoms: dielectric liquids (water, saline solution, glycerol) for homogeneous phantoms; dielectric gels (gelatin, agar) for homogeneous or heterogeneous phantoms; and dielectric solids (plastics, ceramics), which can be used to produce heterogeneous phantoms.It has been found that phantom manufacturing is carried out using three techniques: casting, in which the dielectric material is melted and poured into a mold with the desired shape; 3D printing; and CNC machining.

[0012] More specifically, focusing on the most recent advances in the field, Mashal et al. (Mashal, A., et al. (2011). Microwave and Optical Technology Letters, 53(8), 1896-1902) presented heterogeneous anthropomorphic breast phantoms, based on oil dispersions in gelatin, to create materials that simulate tissues with different adipose compositions for IM. However, these phantoms only represent healthy tissue conditions that mimic different breast densities (percentage of adipose / glandular tissue) and have a regular conical shape. Y. He et al. (He, Y. et al (2019). 3D-printed breast phantom for multi-purpose and multi-modality imaging. Quantitative Imaging in Medicine and Surgery, 9(1), 63-74) used 3D printing to produce multi-purpose and multimodal phantoms (X-ray, US, MRI) based on materials that simulate polyvinyl chloride (PVC) tissues.This set of phantoms was produced for image quality control using controlled, homogeneous internal shapes. Limitations were found in evaluating millimeter-sized lesions, and bubbles appeared in the phantoms due to the PVC mixture. Furthermore, they were unsuitable for microwave imaging applications. Ruvio et al. (Ruvio, G. et al. Sensors, 20(8), 2400) presented multimodal breast phantoms for microwave imaging, ultrasound, mammography, magnetic resonance imaging, and computed tomography. They used material casting and 3D printing to provide different tissue molds; they then used different materials (polyvinyl alcohol cryogel, agar, glycerol, etc.) to represent complex five-layer phantoms composed of pectoral muscle, skin, adipose tissue, fibroglandular tissue, and carcinoma.Furthermore, a patent on the use of liquid solutions to emulate the dielectric behavior of breast tissue has been found in the literature (KR101026833).

[0013] In general, there is a notable lack of research using phantoms of this type. This scarcity is mainly due to the innovative nature of the technology, and to date, there are no commercial offerings for such devices.

[0014] Therefore, to overcome the shortcomings of the state of the art, the present invention presents a methodology for synthesizing specific materials that simulate breast tissue, including materials that simulate tumors, which can be used to manufacture a customizable anthropomorphic phantom with controlled dielectric and mechanical properties.

[0015] DESCRIPTION OF THE INVENTION The female breast is an organ composed of a mixture of different tissues (adipose, fibroglandular), which varies from woman to woman depending on factors such as age, day of the menstrual cycle, and other conditions (e.g., pregnancy, breast implants). According to current medical imaging technologies, tissue properties such as acoustic attenuation (ultrasound), magnetic resonance relaxometry (magnetic resonance imaging), X-ray attenuation (mammography), and dielectric parameters (microwave imaging) must be considered when designing an imaging phantom according to its end applications.

[0016] For this invention, the dielectric properties of breast tissue are of paramount importance, as they will define the corresponding reconstructed microwave image.

[0017] The inventors have discovered that the compound 2-hydroxyethyl acrylate (2-HEA) could be used as the main tissue-simulating material, representing the common dielectric properties of a mixture of adipose and fibroglandular breast tissues, while a modified hyaluronic acid (hyaluronic acid metachlate or MHA) was used to simulate the dielectric properties of common malignant breast tissues.

[0018] Therefore, in the present invention, 2-hydroxyethyl acrylate (2-HEA) is used to prepare a hydrogel that simulates adipose and fibroglandular breast tissue, while hyaluronic acid methacrylate (MHA) is used to prepare a hydrogel that simulates malignant tissue (cancer). The hydrogel formed from 2-HEA (also called 2-HEA hydrogel) comprises cross-linked 2-hydroxyethyl acrylate. The hydrogel formed from the aforementioned MHA (also called HAMA, or hyaluronic acid methacrylate) comprises cross-linked hyaluronic acid methacrylate.

[0019] Furthermore, the composition that mimics breast tissue is 3D printable, allowing it to be printed in a specific shape and size to simulate a real breast.

[0020] Therefore, the present invention relates to a microwave-focused breast phantom made by 3D printing and assembling materials that simulate the dielectric properties of common breast tissue. This phantom can be used by researchers and manufacturers of microwave imaging devices to improve their imaging methods, and by healthcare professionals to learn microwave breast imaging procedures or for other activities requiring a realistic breast phantom.

[0021] Therefore, a first aspect of the present invention relates to a breast phantom comprising:

[0022] - a first hydrogel that forms a simulated normal breast tissue phantom and

[0023] - a second hydrogel forming a tissue phantom simulating breast cancer, wherein the first hydrogel comprises cross-linked 2-hydroxyethyl acrylate, and said first hydrogel is obtained by a 3D printing process comprising: printing a solution comprising 2-hydroxyethyl acrylate (2-HEA), a cross-linking agent and a photopolymerization initiator in a mixture of water and ethanol, wherein the printer employs ultraviolet light that initiates the formation of radicals from the polymerization initiator, thereby polymerizing the mixture and obtaining the breast tissue phantom, and wherein the second hydrogel comprises cross-linked hyaluronic acid methacrylate and said second hydrogel is obtained by introducing an aqueous solution of hyaluronic acid methacrylate (MHA) and a photopolymerization initiator into the structure of the previously prepared breast tissue phantom, preferably through a syringe,Ultraviolet radiation is applied to induce the formation of the hydrogel that makes up the simulated cancerous tissue phantom.

[0024] The term "hydrogel" or "hydrogel composition" refers to a cross-linked polymeric material that is insoluble in water and contains water within its polymer matrix. The hydrogel of the present invention has a solid consistency. Solid hydrogels possess a robust cross-linked network structure, stabilized by covalent crosslinking agents. Although they remain solid at room temperature, they have the ability to swell in water. The hydrogel of the invention has Young's modulus values ​​similar to those of real tissues, between 1 and 200 kPa, more preferably between 10 and 100 kPa, measured from a stress-strain curve using a Mecmesin Multitest 2.5-ia 25°C dynamic mechanical analyzer.

[0025] The term "crosslinking agent" refers to a molecule capable of forming a covalent bond between polymers or between two different regions of the same polymer. The term "photopolymerization initiator" denotes a compound that generates the polymerization factor (radical) in response to light of a predetermined wavelength.

[0026] The 3D printing technique used to obtain the first hydrogel is stereolithography (SLA). Therefore, any stereolithography 3D printer could be used, for example, the Photon M3 Max 3D printer (Anycubic, Shenzhen, China).

[0027] The first hydrogel is printed in the shape and size of a real breast, preferably based on a previously created 3D model.

[0028] To produce efficient models that incorporate specific shapes for 3D printing, anthropomorphic three-dimensional models can be designed prior to printing to obtain hydrogel with a shape and size similar to a real breast. Three-dimensional models based on actual medical breast images can be designed using open-source software for medical image processing and computer-aided design (CAD).

[0029] In a preferred embodiment, in the solution to be printed (to form the first hydrogel), the crosslinking agent is polyethylene glycol diacrylate (PEGDA), preferably PEGDA with an average Mn of 700 g / mol. The use of this particular crosslinker will impart elasticity to the chains (it is a fairly large crosslinker) and provide consistency to the structure so that it can better withstand the fatigue of use.

[0030] In a preferred embodiment, the photopolymerization initiator in the solution to be printed is lithium phenyl-(2,4,6-trimethylbenzoyl) phosphinate (LiTPO). This initiator has its activation peak close to the wavelength used by the 3D printer, making it an ideal candidate. Furthermore, it has been shown to be biocompatible.

[0031] The ethanol-water mixture in the solution to be printed preferably comprises ethanol-water in a volume ratio of 1:1.

[0032] In a preferred embodiment, the concentration of 2-HEA in the solution is between 200 and 1000 mg / mL. In a preferred embodiment, the amount of crosslinking agent in the solution is between 1 and 6 mg / mL.

[0033] In another preferred embodiment, the amount of photopolymerization initiator in the solution is between 1 and 10 mg / mL.

[0034] In a preferred embodiment, the printer employs LEDs that emit light and / or at a preferred wavelength between 365 nm and 405 nm, more preferably 405 nm.

[0035] Regarding the second hydrogel, the aqueous MHA solution and a photopolymerization initiator may also contain a dye, preferably a non-toxic food coloring, such as red food coloring. The dye enhances the visibility of the lesion.

[0036] MHA can be synthesized by any method known in the state of the art, such as the method described by Zhang et al. in 2020 (Zhang, Q. et al. (2020) Journal of Materials Chemistry B, 8(25), 5441-5450), based on the reaction between sodium hyaluronate and methacrylic anhydride (see examples of the present invention).

[0037] The photopolymerization initiator used to prepare the second hydrogel is sodium phenyl-(2,4,6-trimethylbenzoyl) phosphinate (NaTPO).

[0038] In a preferred embodiment, the concentration of MHA in the solution for preparing the second hydrogel is between 8 and 50 mg / mL. Preferably, the amount of photopolymerization initiator in the solution for preparing the second hydrogel is between 0.5 and 5 mg / mL.

[0039] In a preferred embodiment, to prepare the second hydrogel, ultraviolet radiation of 365 nm is applied.

[0040] Preferably, the ultraviolet radiation used to prepare the second hydrogel is applied for a duration of 2-10 minutes.

[0041] The specific reagents and proportions used in the polymerization reaction have yielded hydrogels that mimic the dielectric properties of both normal breast tissue and pathological tissue (breast cancer). These outstanding characteristics of the hydrogels make the phantom suitable for use in microwave imaging (MI), which requires the tissue to have dielectric properties similar to those of the actual organs being analyzed.

[0042] In the present invention, the term "dielectric properties" refers to properties related to electrical conductivity (Ω) [S / m] and permittivity (Ω').

[0043] The first hydrogel preferably has an electrical conductivity of 0.73 ± 0.01 and a permittivity of 27.93 ± 0.91, measured at 4 GHz and 25°C.

[0044] The second hydrogel preferentially has an electrical conductivity of 3.15 ± 0.21 and a permittivity of 73.70 ± 0.96 at 4 GHz and 25°C. Both the electrical conductivity and permittivity were measured with a new open-ended coaxial probe developed by the Microwave and Radiation Laboratory of the University of Pisa, using the Virtual Line Transmission Model (VLTM).

[0045] The hydrogels used in this report are extremely suitable materials because they have demonstrated ideal characteristics for a breast phantom dedicated to microwave imaging. Furthermore, hydrogels can be printed in any shape and size, can be reused multiple times without losing their properties, and possess the mechanical properties and shape of a real breast.

[0046] A second aspect of the invention relates to a process for manufacturing the breast phantom described in the first aspect, wherein the process comprises: a) preparing a first hydrogel by imprinting a solution as described in the first aspect of the invention into the shape and size of a breast to obtain a breast tissue phantom, b) introducing an aqueous solution of MHA and a polymerization initiator into the breast tissue phantom structure obtained in step a) and applying UV radiation to induce the formation of a second hydrogel, as described in the first aspect of the invention.

[0047] To produce efficient models that include specific shapes for 3D printing, preferably, prior to printing, anthropomorphic three-dimensional models can be designed to obtain the hydrogel with a shape and size similar to a real breast, as indicated in the first aspect of the invention. A third aspect of the invention relates to the use of the product described in the first aspect of the invention as a phantom in microwave imaging inspection.

[0048] In a preferred embodiment, the breast phantom is used to test the reliability of a microwave imaging device and / or to calibrate, validate, and optimize microwave imaging devices.

[0049] In another preferred embodiment, the breast phantom is used to conduct research and development in the field of microwave medical imaging.

[0050] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by persons skilled in the field to which this invention pertains. Similar or equivalent methods and materials to those described herein may be used in the practice of the present invention. Throughout the description and claims, the word "comprising" and its variations are not intended to exclude other technical features, additives, components, or steps. Other objects, advantages, and features of the invention will become apparent to those skilled in the art upon examination of the description or may be learned by the practice of the invention. The following examples and drawings are provided for illustrative purposes and are not intended to limit the scope of the present invention.

[0051] BRIEF DESCRIPTION OF THE DRAWINGS

[0052] FIG. 1. (a) Schematic of the synthesis and 3D printing of 2-HEA; (b) Schematic of the synthesis of HAMA.

[0053] FIG. 2. Stress-strain curves and Young's modulus for 2-HEA and HAMA hydrogels.

[0054] FIG. 3. Stress-time curves for viscoelasticity tests: (a) 2-HEA hydrogels, (b) HAMA hydrogels.

[0055] FIG. 4. Stress-strain curve for tensile tests of the 2-HEA hydrogel.

[0056] FIG. 5. Cyclic fatigue test for the 2-HEA hydrogel.

[0057] FIG. 6. SEM scan: HAMA.

[0058] FIG. 7. Pore size distribution of HAMA hydrogel.

[0059] FIG. 8. SEM images of the bond between the 2-HEA hydrogel and HAMA. FIG. 9. Infrared spectrum of (a) 2-HEA hydrogel, (b) 2-HEA monomer, (c) HAMA hydrogel.

[0060] FIG. 10. Dielectric properties: complex permittivity (left) and conductivity (right) of several 2-HEA hydrogel samples. Theoretical values ​​used as a reference are also shown.

[0061] FIG. 11. Dielectric properties: complex permittivity (left) and conductivity (right) of two HAMA hydrogel samples. Theoretical values ​​used as a reference are also shown.

[0062] FIG. 12. Example of microwave acquisition without test sample (empty device).

[0063] FIG. 13. Example of microwave acquisition of the phantom prototype with reference to 0°

[0064] FIG. 14. Image of the post-processed 3D model that includes an irregular and malignant lesion.

[0065] EXAMPLES

[0066] The following examples are given to illustrate the invention, but are not intended to be limiting.

[0067] 1. Preparation of a mammary phantom of the invention

[0068] 1.1. Design of three-dimensional anthropomorphic models

[0069] 3D models can be designed using open-source software for medical image processing and computer-aided design (CAD). 3D Slicer (v. 5.2.2) on macOS (Ventura 13.2.1) was used to generate accurate 3D breast models based on actual medical images. Reference MRI images were extracted from The Cancer Imaging Archive (TCIA), which hosts a large archive of labeled cancer medical images accessible for public download. For our purposes, the Breast-Diagnosis collection was downloaded, as it contains numerous examples of both normal and cancerous breasts. Magnetic resonance imaging was then used to extract breast morphological features and tumor anatomy to create the 3D models.Various segmentation approaches were tested and used to ensure the best resolution, including threshold-based segmentation (Otsu, Huang, IsoData, Kittier-Illingworth, maximum entropy, moments, Renyi entropy, Shanbhag, Triangle, Yen) and level tracing. To produce efficient models that include specific shapes for 3D printing, contour and lesion-independent segmentation was performed, followed by subtraction in 3D Slicer. The generated 3D models needed post-processing to remove unwanted structures from the segmentation and provide contours suitable for 3D printing. Autodesk Meshmixer was then used for post-processing the models on macOS (Ventura 13.2.1). This method worked well and provided efficient models while also outputting print-ready 3D designs.

[0070] Specifically, the morphological characteristics of the proposed phantom were extracted using magnetic resonance imaging from the BreastDX-01-0067 sample in the aforementioned database. The model represented the breast of a 70-year-old woman with confirmed breast cancer in the left breast. This breast cancer was associated with a 2.8 x 2.3 x 2.4 cm mass in the upper outer region, located 10 cm from the nipple and 2.2 cm from the chest wall. The mass was reported to be an invasive ductal carcinoma.

[0071] The corresponding MRI sequences from BreastDX-01-0067 were imported in DICOM format into 3D Slicer. Three different MRI sequences are provided (before and after contrast administration to the patient): T2W_TSE SENSE, STIR SENSE, and AX BLISS AUTO SENSE. The STIR sequence was used for segmentation because it provides good resolution and definition of the breast contour and lesion margins due to its fat suppression algorithm for image reconstruction. First, a threshold (between 609.22 and 10412.5) was used to define the model containing the volumetric information of both breasts with their corresponding defined shape (this operation was called segmentation). Then, the left breast and other irrelevant body parts were removed.Next, processing was performed on the various steps of the MRI sequence to optimize segmentation 1 by deleting unwanted pixels outside the breast contour and filling empty pixels within the volumetric model. The corresponding lesion was then segmented (this operation was noted as segmentation 2). Level tracing was used on the various steps of the MRI sequence to define and include the entire lesion area as part of the segmented area. Finally, automatic pixel subtraction was performed between segmentations 1 and 2 to obtain a rendered 3D model with the optimal breast shape, including a gap with the dimensions and irregular shape of the corresponding tumor lesion. 1.2. Synthesis of materials, fabrication, and assembly of the system.

[0072] As previously mentioned, microwave imaging methods rely on the dielectric behavior of materials (i.e., their dielectric properties). Thus, to mimic the dielectric conductivity and permittivity of breast tissue, the inventors discovered that the 2-HEA compound could be used as the primary tissue-mimicking material, representing the common dielectric properties of a mixture of adipose and fibroglandular breast tissue. Meanwhile, a modified MHA was used to form the HAMA hydrogel, which mimics the dielectric properties of common malignant breast tissue. Figure 1 illustrates the overall synthesis process for both hydrogel-based materials.

[0073] The preparation of the 2-HEA hydrogel phantoms was carried out using the Photon M3 Max 3D printer (Anycubic, Shenzhen, China), which can print the specific .sí / model file. This printer operates on a bottom-up principle, whereby the model is projected onto the container base through a transparent membrane. The printing material consists of a solution composed of 2-HEA, polyethylene glycol diacrylate (PEGDA), and lithium phenyl-(2,4,6-trimethylbenzoyl)phosphinate (LiTPO) in a mixture of MilliQ water and ethanol with a 1:1 volume ratio. Specifically, the mixture consisted of 200–1000 mg / ml of 2-HEA, 1–6 mg / ml of PEGDA, and 1–10 mg / ml of LiTPO in the water and ethanol mixture. The 2-HEA was purchased from TCI Chemicals. The PEGDA (Mn 700) and LiTPO were purchased from Sigma-Aldrich and used as received. Absolute ethanol was purchased from Labkem.After dissolving the mixture in the solvent with stirring, it was introduced into the printing container. For polymerization, the printer uses LEDs that emit light at a wavelength of 405 nm, which initiates the formation of radicals from the LiTPO molecule. The optimal printing parameters used in the synthesis of 2-HEA hydrogel-based phantoms were: Layer thickness of 0.05–0.2 mm, normal exposure time of 15–35 s, power-off time of 1 s, bottom exposure time of 40–60 s, and 6 bottom layers. Z-lift distance was set to 6 mm, and Z-lift speed and Z-refractive speed were both set to 6 mm / s.

[0074] After creating the simulated breast tissue phantom, a hyaluronic acid hydrogel precursor solution (composed of 8–50 mg / mL of MHA and 0.5–5 mg / mL of NaTPO using MilliQ water as the solvent) is introduced into the structure using a syringe. This precursor solution consists of MHA, along with NaTPO, which serves as a photochemical initiator; red dye was also included to enhance the visibility of the lesion. Subsequently, a 365 nm UV lamp is applied to the phantom silhouette for 2–10 minutes to induce hydrogel formation, which mimics a tumor-like structure.

[0075] The MHA was synthesized based on the method described by Zhang et al. in 2020 (Zhang, Q. et al. (2020). Journal of Materials Chemistry B, 8(25), 5441-5450). The procedure involves dissolving 2 grams of sodium hyaluronate in 100 mL of MilliQ water under constant stirring. Subsequently, 67 mL of dimethylformamide (DMF) are added, and the solution is cooled to 4°C. 1.54 grams of methacrylic anhydride are added dropwise at a rate of 1 drop per second while maintaining the pH between 8 and 9 for 4 hours using 0.5 M NaOH. The mixture is then stirred continuously at 4°C for 12 hours, after which 4.88 grams of NaCl are added. After the NaCl has dissolved, 200 mL of ethanol are added as a precipitant. The precipitate is washed with mixtures of water and ethanol (3 / 7, 1 / 4, and 1 / 9, respectively). Once the supernatant is removed, the precipitate is dissolved in 100 mL of MilliQ water and dialyzed for 3 days.Finally, the solution is lyophilized to obtain hyaluronic acid methacrylate. Sodium chloride (NaCl) was purchased from Sigma-Aldhch and used as received, N,N-dimethylformamide (DMF) was purchased from PanReac AppliChem, and sodium hyaluronate from Guinama. Sodium phenyl-(2,4,6-trimethylbenzoyl) phosphinate (NaTPO) was acquired from Molbase, and methacrylic anhydride from TCI Chemicals.

[0076] 1.3. Characterization of the phantom materials

[0077] Female breasts are composed of a mixture of different tissues (adipose, fibroglandular), the composition of which varies from woman to woman depending on factors such as age, day of the menstrual cycle, and other conditions (e.g., pregnancy, breast implants). According to current imaging technologies, tissue properties such as acoustic attenuation (ultrasound), MRI relaxometry (magnetic resonance imaging), X-ray attenuation (mammography), or dielectric parameters (microwave imaging) must be considered when designing an imaging phantom for its intended applications. For this invention, the dielectric properties of the breasts are of paramount importance, as they will define the corresponding reconstructed microwave image.Although not representative of current imaging technologies, the biomechanical properties of breast tissue have been extensively studied, and some research suggests that understanding tissue biomechanical behavior can lead to new imaging techniques, as tissue biomechanical properties change in the presence of cancer. Therefore, to provide a detailed characterization of the materials, their mechanical properties have been evaluated, as it is also crucial that users experience a tactile sensation similar to that of real organs.

[0078] Mechanical characterization

[0079] To assess the elasticity of the synthesized hydrogels, their mechanical properties were examined using compression tests. The Young's modulus was calculated from stress-strain curves obtained at an initial degree of swelling in water at room temperature. Cylindrical hydrogel samples were then compressed between two plates. The Young's modulus (Figure 2) was calculated based on the linear segment of the curves (corresponding to 2–10% strain). For the 2-HEA hydrogels, the Young's modulus was determined to be 44.8 ± 8.0 kPa, while for the hyaluronic acid-derived hydrogels, the measured Young's modulus was 5.8 ± 1.2 kPa. These results suggest that the synthetic hydrogels exhibit greater workability and improved elasticity compared to the hyaluronic acid hydrogel.

[0080] Viscoelastic studies were performed to evaluate the tension relaxation of the hydrogels. The viscoelasticity of the hydrogels was measured by subjecting them to compressions of 15% and 40% of their deformation and monitoring their tension for a period of 10 minutes (Figure 3). In this respect, hyaluronic acid hydrogels showed lower relaxation values ​​than synthetic hydrogels, at both 15% and 40% compression.

[0081] Next, the ultimate tensile strength was calculated. Six balanced dumbbell samples, also known as dog bone samples, were prepared. These samples had an effective length of 20 mm and a cross-section of 4 x 2 mm. 2 , dimensions closely aligned with ISO 37 (ISO 37:2017. Rubber, vulcanized or thermoplastic - Determination of tensile stress-strain properties).

[0082] The samples were subjected to uniaxial stretching at a speed of 50 mm / min until they reached their breaking point. During this test, data were obtained on the tensile strength (TS), which represents the maximum stress the material can withstand before breaking, and the strain at failure (SAF), which indicates the amount of deformation or elongation at the point of failure. In addition, the fracture toughness of the material under an applied load was calculated. Fracture toughness is a measure of the material's ability to resist the propagation of cracks or fractures when subjected to tension and is a crucial parameter for evaluating its durability and reliability in practical applications. Specifically, for the 2-HEA hydrogel, a TSS of 31.6 ± 5.1 kPa, an SAF of 2.2 ± 0.2, and a fracture toughness of 39.5 ± 8.3 kJ / m³ were obtained.The HAMA hydrogel has not been subjected to uniaxial stretching, as it will be contained within the 2-HEA phantom body; therefore, HAMA will not experience any kind of tension.

[0083] Finally, the use of these phantoms may require many movements during their life cycle (since these phantoms can have different applications), so it is necessary to study how the material behaves under various repeated deformations. Therefore, fatigue tests are performed, consisting of many compression cycles carried out at a speed of 30 mm / min. Each cycle ranges from compression from 0% to 40% displacement and relaxation from 40% to 0% displacement. The cylindrical samples have dimensions of 5 mm in height and 10 mm in diameter. Fatigue tests are performed to achieve 100 compression cycles on each hydrogel. As can be seen in Figure 5, the maximum stress value of the first cycle reaches 52.9 ± 4.4 kPa and decreases to 46.1 ± 1.9 kPa, representing a 13% reduction from its maximum stress value.As in the tensile tests, in the fatigue tests only the 2-HEA hydrogels were measured, since the HAMA hydrogel will be on the inside and therefore will not be subjected to tension.

[0084] Scanning electron microscopy

[0085] The pore morphology of the hydrogels was evaluated by scanning electron microscopy (SEM) using a Gemini SEM 500 (Zeiss, Jena, Germany) integrated with an FEI QUANTA 250 module. The hydrogels were expanded to their maximum swelling state. To ensure complete freezing, the samples were rapidly frozen in liquid nitrogen for a period of time. The resulting frozen hydrogels were lyophilized overnight to obtain dry aerogel samples.

[0086] The pore size distribution was analyzed at maximum swelling in MilliQ water. In this case, the pore size could only be determined for the hyaluronic acid-derived hydrogel (Figure 6) because the 2-HEA hydrogel did not have visible pores in its structure. The pore size of the HAMA hydrogel was 65.4 ± 1.6 µm (Figure 7). Furthermore, the bond between the two hydrogels was studied using this technique, as shown in Figure 8.

[0087] Fourier Transform Infrared Spectroscopy

[0088] Figure 9a shows the vibrational bands associated with the synthesized 2-HEA hydrogel. In particular, the distinctive carbonyl group stretching band is observed at 1720 cm⁻¹ -1 The CH stretching band is located at 2955 cm -1 , while the CH2 band appears at 1448 erm 1 The OH stretching band is prominently featured at 3397. 1, followed by the OH flex band at 1254 crm 1 Furthermore, the stretching band attributed to the C-0 group of the ester is manifested at 1163 cm 1 Interestingly, the bands located at 1628 and 980 crm 1 The bands corresponding to the C=C bond present in the 2-HEA monomer (Figure 9b) are completely absent in the hydrogel spectra. This absence clearly suggests the completion of the polymerization process. Furthermore, comparing both spectra in Figure 9, we can see how the bands related to the OH bond (OH stretching and C-O stretching of the ester group) shift. Meanwhile, the remaining bands (CH stretching and C=O stretching) not related to this bond do not shift. This may be due to the formation of hydrogen bonds in the hydrogel.

[0089] The HAMA hydrogels were also subjected to infrared spectroscopy (Figure 9c). Around 3330 cm -1A broad band characteristic of the OH and NH stretching of the acetyl group appears. The CH segment band appears at 2920 cm⁻¹ -1 The bands at 1609 and 1375 cm -1 These effects are due to the asymmetric (C=O) and symmetric (CO) stretching modes of the hyaluronate carboxyl groups. The absorption band at 1038 cm -1 This is due to the cyclic stretching of the ether (COC).

[0090] Dielectric characterization

[0091] The dielectric characterization of the materials used was performed using an open coaxial probe developed by a spin-off company of the University of Pisa (Free Space Srl, Pisa, Italy). This method, which employs the Virtual Line Transmission Model (VLMT), has been presented, tested, and verified with various substances, materials, and biological samples. It enabled the instantaneous and non-destructive characterization of soft solid, liquid, and semi-solid materials with a simple setup consisting of a truncated section of coaxial cable connected to a vector network analyzer (Copper Mountain Technologies, Indianapolis, USA). Using this technique, reflection coefficients (Su) were acquired and processed to reconstruct the dielectric properties of the materials in terms of complex permittivity and conductivity across a variable frequency range (1 to 9 GHz).The model presented and experimentally validated by Sugitani et al. was used as reference values ​​for conductivity and permittivity in breast tissue (Sugitani et al. (2014). Applied Physics Letters, 104(25), 253702). Table 1 presents the dielectric properties, encompassing both electrical conductivity and the real part of the permittivity, for the theoretical model used and the experimentally reconstructed values ​​of the native (newly synthesized) and modified HAMA materials. Figure 11 shows several experimental dielectric parameters within the frequency range of 0.5 to 9 GHz (this frequency range was selected based on that used by a commercial microwave imaging system provider—the MammoWave system, UBT srl, Italy—which will be used for phantom validation, with the theoretical model values ​​also included for comparison).

[0092] Table 1. Dielectric properties at 4 GHz, including the theoretical values ​​(Sugitani et al., 2014) used as a reference, and the experimental values ​​of the soft materials used Applicability validation

[0093] The presented invention has been tested and validated on a commercial microwave breast imaging device, MammoWave (UBT srl, Italy). This system has already been tested with canonical phantoms and human volunteers in clinical studies. The device consists of two antennas (one transmitting and one receiving), connected to a 2-port VNA that generates and collects scattered electromagnetic signals. The device uses 10 transmission positions (grouped into five double positions centered at 0). o, 72°, 144°, 216° and 288°, respectively) for each of which, the receiving antenna rotates 360° around the object under test, collecting the signals at 80 different positions with a step of 4.5°. The scattered electromagnetic signals are then processed by a specific imaging algorithm based on the Huygens principle (Enders, P. (1996). European Journal of Physics, 17(4), 226-235), which produces an intensity map of the dielectric properties of the object under test.

[0094] To verify MammoWave's ability to detect the simulated tumor in the presented invention, several tests were performed under controlled conditions (temperature, humidity) by scanning the proposed phantom at different angular positions (the phantom was placed in various positions to analyze potential variations in signal emission and transmission in the prototype). A reference line was included on the phantom to control its position on top of the MammoWave scanning chamber. Image homogeneity was then measured using several conductivity-weighted image parameters—that is, features that can provide an indication of dielectric inhomogeneity when compared to reference lines, such as images of the empty device. The microwave images were reconstructed using a conductivity value of 0.3 S / m.

[0095] Specifically, the following features were extracted from the reconstructed MammoWave images, as determined in previous studies: max_n (maximum intensity value of the normalized image), max2mean_i (maximum intensity value of the image in the full domain / mean value of the image in the full domain), var_p (image vahence in the peak region), mad0_p (mean absolute deviation of the image in the peak region), and var_r (ratio of image vahence in the peak region to its complementary region). The presence of the mimicking lesion was assessed using the reference features from a set of control acquisitions. The calculated characteristics of the images used as a reference were the following: max_n of 0.0463 ± 0.0003, max2mean_¡ of 1.4684 ± 0.0119, var_p of 0.0022 ± 0.0005, mad_p of 0.0275 ± 0.0003, and var_r of 0.0254 ± 0.0056.Figure 12 shows an example of a conductivity-weighted microwave image (0.3 S / m) with the chamber empty (used as a reference).

[0096] Figure 13 provides an example of a conductivity-weighted microwave image of the proposed invention at an angular position of 0 o Note that the black line refers to the reference line used for positioning the phantom on top of the MammoWave acquisition chamber, while the arrow refers to the peak associated with the mimic inclusion. It can be observed that HAMA can be visually represented due to its higher dielectric properties compared to the surrounding material (2-HEA). Note that phantom peaks, related to the image artifact, can also be observed. Finally, the image characteristics allowed for the detection of lesions mimicking tumors.

[0097] 3. Conditions for the preservation and use of phantoms

[0098] Water plays a crucial role in determining the properties and structures of hydrogels. Fluctuations in the water content of hydrogels due to variations in temperature or humidity can affect their dielectric properties and, consequently, the applicability of the proposed phantoms (Pissis et al., 2000). Therefore, we investigated this impact through two main tests on prototype vapors of the proposed phantom.

[0099] 3.1. Usability testing of the prototype

[0100] The aim of this set of experiments is to determine whether the proposed phantom changes its properties when handled for its intended use. Specifically, we have two objectives: 1) to determine whether the external 2-HEA material of the phantom changes its dielectric properties when handled and stored for several working days, and 2) whether there is a gradual loss of dielectric mismatch between the 2-HEA and HAMA materials when the phantom is used under working conditions.

[0101] First, a prototype phantom (total mass 262.3 g; mass of the phantom without HAMA inclusion: 261.9 g) was dielectrically characterized over seven days. Specifically, the phantom was stored in a sealed glass container under controlled conditions (ambient temperature of 22°C and 50% humidity). The phantom was manipulated daily, and dielectric characterization was performed (always at the same points on the surface) to obtain the dielectric parameters of the external 2-HEA (conductivity and permittivity). The phantom was also weighed to measure water loss. The results are presented in Table 2. Table 2. Mass, conductivity, and permittivity of the 4 GHz 2-HEA prototype phantom

[0102] Next, batch tests were performed using microwave imaging of the phantom prototype, also on day 1, using the MammoWave device. Six microwave acquisitions were taken as a baseline with the device without any sample. Then, 13 subsequent microwave acquisitions (i.e., referring to approximately 3 hours of phantom handling under working conditions) were performed with the phantom in a fixed position, at 0 oAt ambient temperature and humidity (22°C and 50%), microwave images were reconstructed at 0.3 S / m, extracting feature lines for each image. Dielectric changes in both materials (2-HEA, HAMA) were measured using these features, allowing for a quantitative description of the material mismatch. The analysis, shown in Table 3, indicated significant dielectric changes, suggesting the presence of the simulated tumor. Finally, a comparative analysis of the initial and final acquisitions confirmed the stability of the dielectric properties throughout the testing process, indicating that the dielectric mismatch between the materials remained constant. A maximum percentage increase of 5% was obtained in the Max_n parameter (2.7%, 2.9%, and 2.1% reductions for Max2Avg, Var_p, and Var_r, respectively, with a 0.09% reduction for Mad_p). Table 3.Microwave imaging parameters: reference values ​​(empty device) versus proposed phantom scan.

[0103] Recovery to the initial state

[0104] To verify whether the fabricated state of the phantom could be recovered after 7 days (in terms of total mass and dielectric properties of 2-HEA) in the prototype used for the previous experiments, the phantom was stabilized for 48 hours in a sealed container within a distilled water medium (with the aim of naturally recovering the initial water content). Following this process, the external surface of the 2-HEA phantom was dielectrically characterized, and the mass of the prototype was also measured.

[0105] For further details, two weeks after the manufacturing date, the phantom's mass was 245.2 g (a 6.5% mass reduction). The phantom was then submerged in 17.1 g of distilled water (mass loss relative to its original manufacturing state) and stored in a completely sealed environment for 48 hours to allow the prototype to reabsorb the water and regain its original weight and properties. After this period, the phantom's mass reached 256.8 g, and its 2-HEA dielectric properties at 4 GHz were as follows: conductivity of 0.65 ± 0.03 S / m and permittivity of 22.57 ± 1.93, which match the phantom's property values ​​immediately after manufacturing. 3.2. Prototype Preservation Tests

[0106] The objective of this set of experiments is to determine whether the proposed phantom changes its condition when stored for a defined period (14 days) in a sealed glass container within a controlled environment (ambient temperature of 22°C and humidity of 50%). To this end, the experiment will consist of two main tests performed after the phantom's fabrication (total mass of 272.3 g; mass of the phantom without HAMA inclusion: 271.9 g) (day 1) and after the 14-day sealed storage period: 1) Performing the dielectric characterization of the external 2-HEA hydrogel to measure its dielectric properties; and 2) Using microwave imaging with the MammoWave device to determine the dielectric mismatch between the 2-HEA and the modified HAMA (effects of water loss over time) based on the image characteristics.Twenty microwave imaging tests were performed on different phantom configurations (lasting approximately 4 hours); the phantom mass was collected before and after each test. No significant differences were found in the dielectric properties of the prototype's external 2-HEA on day 1 and day 14, although a 6.3% mass reduction was observed. Table 4 provides information on the mass and dielectric characteristics of the external 2-HEA surface on day 1 and day 14.

[0107] Table 4. Mass, conductivity, and permittivity at 4 GHz of the 2-HEA phantom prototype after phantom fabrication and after 14 days of storage

[0108] Finally, to better understand the dielectric properties of the 2-HEA-HAMA complex and define the specific applicability conditions and optimal storage of the phantom, it is necessary to perform specific dielectric relaxation spectroscopy measurements at different temperatures and humidity conditions for a fixed water content (i.e., according to the recipe). Longer storage times, higher temperatures, or varying humidity conditions can affect the degree of HAMA swelling and water transfer with the surrounding 2-HEA gel.However, if such a loss of the HAMA structure occurs, the phantom can be easily reassembled by reintroducing the corresponding solution and inducing the polymerization process, while the total loss of water from the phantom can be fixed by stabilizing the phantom prototype in a tank containing distilled water (specifically adding the lost mass).

Claims

CLAIMS 1. A breast phantom, comprising: - a first hydrogel that forms a simulated phantom of breast tissue and - a second hydrogel forming a simulated phantom of cancerous breast tissue, wherein the first hydrogel comprises crosslinked 2-hydroxyethyl acrylate and said first hydrogel is obtained by a 3D printing process comprising: printing a solution containing 2-hydroxyethyl acrylate (2-HEA), a crosslinking agent and a photopolymerization initiator in a mixture of water and ethanol, wherein the printer uses ultraviolet (UV) light that initiates the formation of radicals from the polymerization initiator, thereby polymerizing the mixture and obtaining the breast tissue phantom, and wherein the second hydrogel comprises crosslinked hyaluronic acid methacrylate and said second hydrogel is obtained by introducing an aqueous solution of hyaluronic acid methacrylate (MHA) and a photopolymerization initiator into the structure of the previously prepared breast tissue phantom,UV radiation is applied to induce the formation of the hydrogel that makes up the simulated phantom of cancerous tissue.

2. The breast phantom, according to claim 1, wherein the first hydrogel is obtained by stereolithography 3D printing.

3. The breast phantom, according to claim 1 or 2, wherein the crosslinking agent in the solution to be printed is polyethylene glycol diacrylate (PEGDA).

4. The breast phantom, according to any of the preceding claims, wherein the photopolymerization initiator in the solution to be printed is lithium phenyl-(2,4,6-trimethylbenzoyl) phosphinate (LiTPO).

5. The breast phantom, according to any of the preceding claims, wherein the concentration of 2-HEA in the solution is between 200-1000 mg / mL.

6. The breast phantom, according to any of the preceding claims, wherein the concentration of the crosslinking agent in the solution is between 1 and 6 mg / mL and / or the amount of photopolymerization initiator in the solution is between 1 and 10 mg / mL.

7. The breast phantom, according to any of the preceding claims, wherein the printer uses light with a wavelength of 405 nm.

8. The breast phantom, according to any of the preceding claims, wherein the aqueous MHA solution and the photopolymerization initiator also contains a dye.

9. The breast phantom, according to any of the preceding claims, wherein the photopolymerization initiator used to prepare the second hydrogel is sodium phenyl-(2,4,6-trimethylbenzoyl) phosphinate (NaTPO).

10. The breast phantom, according to any of the preceding claims, wherein the concentration of MHA in the solution used to prepare the second hydrogel is between 8 and 50 mg / mL, and preferably, the amount of photopolymerization initiator is between 0.5 and 5 mg / mL.

11. The breast phantom, according to any of the preceding claims, wherein, to prepare the second hydrogel, 365 nm UV radiation is applied.

12. The breast phantom, according to any of the preceding claims, wherein the UV radiation used to prepare the second hydrogel is applied for a duration of 2 to 10 minutes.

13. A process for manufacturing the breast phantom described in any of claims 1 to 12, wherein the process comprises: a) preparing a first hydrogel by printing a solution as described in any of claims 1 to 7, in the shape and size of a breast to obtain a breast tissue phantom, b) introducing an aqueous solution of MHA and a polymerization initiator into the breast tissue phantom structure obtained in step a) and applying UV radiation to induce hydrogel formation, as described in any of claims 1, 8-12.

14. Use of the breast phantom described in any of claims 1 to 12 as a phantom in microwave imaging inspection.

15. Use, according to claim 14, for testing the reliability of a microwave imaging diagnostic device and / or for calibrating, validating, and optimizing microwave imaging diagnostic devices.

16. Use, according to claim 14, for conducting research and development in the field of microwave medical imaging.

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