Design, Development and Validation of a Knee Brace to Standardize the US Imaging Evaluation of Knee Osteoarthritis.

OBJECTIVE: A repeatable and reliable follow-up of knee injuries would be desirable to prevent delayed diagnosis and to monitor the efficacy of the applied treatment over time. Ultrasound (US) techniques are an attractive option to this purpose, since they are safe, low-cost and non-invasive. However, its use in the clinical practice is limited by the high dependency on the operator's experience. Hence, the objective of this study is to provide a standardization of the US image acquisition process for knee osteoarthritis (OA) allowing an extended clinical use of US technologies in this domain. METHODS: Clinical specifications were provided by expert musculoskeletal radiologists thus identifying the subject poses and the US probe positions needed to evaluate the cartilage structure, signs of synovitis and joint effusion. Such considerations were used to derive the technical requirements needed for the development of a wearable brace equipped with specific openings to guide the correct placement of the probe. The feasibility of the developed wearable brace was tested on three healthy volunteers, which were asked to acquire informative US images, similar to the reference images performed by the musculoskeletal radiologist. RESULTS: Thanks to the knee brace, the untrained subjects were able to self-acquire informative B-mode images comparable to the corresponding images acquired by an expert clinician. DISCUSSION/CONCLUSION: The use of a knee brace intended for knee OA US diagnosis demonstrated the possibility to standardize the acquisition protocol and make its application achievable also for untrained subjects, representing a key step toward tele-ultrasonography.

Ex-vivo quantitative ultrasound assessment of cartilage degeneration.

Osteoarthritis is a common disease that implies joint degeneration and that strongly affects the quality of life. Conventional radiography remains currently the most used diagnostic method, even if it allows only an indirect assessment of the articular cartilage and employ the use of ionizing radiations. A non-invasive, continuous and reliable diagnosis is crucial to detect impairments and to improve the treatment outcomes.Quantitative ultrasound techniques have proved to be very useful in providing an objective diagnosis of several soft tissues. In this study, we propose quantitative ultrasound parameters, based on the analysis of radiofrequency data derived from both healthy and osteoarthritis-mimicking (through chemical degradation) ex-vivo cartilage samples. Using a transmission frequency typically employed in the clinical practice (7.5-15 MHz) with an external ultrasound probe, we found results in terms of reflection at the cartilage surface and sample thickness comparable to those reported in the literature by exploiting arthroscopic transducers at high frequency (from 20 to 55 MHz). Moreover, for the first time, we introduce an objective metric based on the phase entropy calculation, able to discriminate the healthy cartilage from the degenerated one.Clinical Relevance- This preliminary study proposes a novel and quantitative method to discriminate healthy from degenerated cartilage. The obtained results pave the way to the use of quantitative ultrasound in the diagnosis and monitoring of knee osteoarthritis.

A novel quantitative and reference-free ultrasound analysis to discriminate different concentrations of bone mineral content.

Bone fracture is a continuous process, during which bone mineral matrix evolves leading to an increase in hydroxyapatite and calcium carbonate content. Currently, no gold standard methods are available for a quantitative assessment of bone fracture healing. Moreover, the available tools do not provide information on bone composition. Whereby, there is a need for objective and non-invasive methods to monitor the evolution of bone mineral content. In general, ultrasound can guarantee a quantitative characterization of tissues. However, previous studies required measurements on reference samples. In this paper we propose a novel and reference-free parameter, based on the entropy of the phase signal calculated from the backscattered data in combination with amplitude information, to also consider absorption and scattering phenomena. The proposed metric was effective in discriminating different hydroxyapatite (from 10 to 50% w/v) and calcium carbonate (from 2 to 6% w/v) concentrations in bone-mimicking phantoms without the need for reference measurements, paving the way to their translational use for the diagnosis of tissue healing. To the best of our knowledge this is the first time that the phase entropy of the backscattered ultrasound signals is exploited for monitoring changes in the mineral content of bone-like materials.

A Reusable Thermochromic Phantom for Testing High Intensity Focused Ultrasound Technologies.

High Intensity Focused Ultrasound (HIFU) surgery is a promising technology for the treatment of several pathologies, including cancer. Testing is a fundamental step for verifying treatment efficacy and safety. Ex-vivo tissues represent the most common solution for replicating the properties of human tissues in the HIFU operative scenario. However, they constitute an avoidable waste of resources. Thus, tissue mimicking phantoms have been investigated as a more sustainable and reliable alternative. In this scenario, we proposed a reusable silicone-based thermochromic phantom. It is cost-effective and can be rapidly fabricated. The acoustic, mechanical, and thermal characterization of the phantom are reported. The phantom usability was evaluated with a HIFU robotic platform. 18 different working conditions were tested by varying both sonication power and duration. Temperature and simulated lesions' size were quantified for all testing conditions. An accordance between temperature and lesion dimension trend over time was found. The proposed phantom results a valid alternative to ex-vivo tissues, especially in the early stages of developing novel HIFU treatment paradigms.

Development and validation of low-intensity pulsed ultrasound systems for highly controlled in vitro cell stimulation.

This work aims to describe the development and validation of two low-intensity pulsed ultrasound stimulation systems able to control the dose delivered to the biological target. Transducer characterization was performed in terms of pressure field shape and intensity, for a high-frequency range (500 kHz to 5 MHz) and for a low-frequency value (38 kHz). This allowed defining the distance, on the beam axis, at which biological samples should be placed during stimulation and to exactly know the intensity at the target. Carefully designed retaining systems were developed, for hosting biological samples. Sealing tests proved their impermeability to external contaminants. The assembly/de-assembly time of the systems resulted ~3 min. Time-domain acoustic simulations allowed to precisely estimate the ultrasound beam within the biological sample chamber, thus enabling the possibility to precisely control the pressure to be transmitted to the biological target, by modulating the transducer's input voltage. Biological in vitro tests were also carried out, demonstrating the sterility of the system and the absence of toxic and inflammatory effects on growing cells after multiple immersions in water, over seven days.

A new safety index based on intrapulse monitoring of ultra-harmonic cavitation during ultrasound-induced blood-brain barrier opening procedures.

Ultrasound-induced blood-brain barrier (BBB) opening using microbubbles is a promising technique for local delivery of therapeutic molecules into the brain. The real-time control of the ultrasound dose delivered through the skull is necessary as the range of pressure for efficient and safe BBB opening is very narrow. Passive cavitation detection (PCD) is a method proposed to monitor the microbubble activity during ultrasound exposure. However, there is still no consensus on a reliable safety indicator able to predict potential damage in the brain. Current approaches for the control of the beam intensity based on PCD employ a full-pulse analysis and may suffer from a lack of sensitivity and poor reaction time. To overcome these limitations, we propose an intra-pulse analysis to monitor the evolution of the frequency content during ultrasound bursts. We hypothesized that the destabilization of microbubbles exposed to a critical level of ultrasound would result in the instantaneous generation of subharmonic and ultra-harmonic components. This specific signature was exploited to define a new sensitive indicator of the safety of the ultrasound protocol. The approach was validated in vivo in rats and non-human primates using a retrospective analysis. Our results demonstrate that intra-pulse monitoring was able to exhibit a sudden appearance of ultra-harmonics during the ultrasound excitation pulse. The repeated detection of such a signature within the excitation pulse was highly correlated with the occurrence of side effects such as hemorrhage and edema. Keeping the acoustic pressure at levels where no such sign of microbubble destabilization occurred resulted in safe BBB openings, as shown by MR images and gross pathology. This new indicator should be more sensitive than conventional full-pulse analysis and can be used to distinguish between potentially harmful and safe ultrasound conditions in the brain with very short reaction time.

Highly controlled and usable system for Low-Intensity Pulsed Ultrasound Stimulation of Cells.

This work aims to describe the design and development of an in vitro highly controlled ultrasonic stimulation system able to guarantee, at the same time, high usability and full sterility of the tested samples. After creating the first prototype of an ultrasound-transparent three-chambers culture well, sealing tests were conducted to prove its impermeability to external contaminants and in vitro tests were carried out to verify the usability of this system for ultrasonic stimulation of cells in vitro. No statistically significant differences were found between control and tested samples during sealing tests, thus demonstrating optimal sealing ability towards external contaminants. Furthermore, the thin polystyrene membrane used to guarantee US-transparency guaranteed a good adhesion and viability of both human fibroblasts and induced pluripotent stem cells.

A Pilot Study for a Quantitative Evaluation of Acoustic Coupling in US-guided Focused Ultrasound Surgery.

In Ultrasound-guided High Intensity Focused Ultrasound (USgHIFU) surgery, the verification of the acoustic coupling correctness between the HIFU transducer and the patient's body is a fundamental step for an efficient and safe therapy. Nowadays, clinicians perform this check by qualitative inspecting Ultrasound images. The aim of this study is the introduction of an objective index to quantitively evaluate the coupling on the base of the radiofrequency echo signals acquired during a low-energy HIFU shot. The experimental session involved a tissue mimicking phantom and a robotic system composed by a HIFU therapeutic transducer and a 2D confocal Ultrasound probe. 15 different coupling conditions between the phantom and the transducer were tested: in each of them, the maximum absolute value of the Fourier Transform of the echo signals was computed and employed to determine an Acoustic Coupling (AC) coefficient.This metrics showed a sigmoidal trend between AC coefficient and coupling increase. This curve can be employed as a calibration tool to quantitatively assess the correctness of the therapeutic set-up before starting the HIFU treatment.

Ex Vivo Assessment of Multiple Parameters in High Intensity Focused Ultrasound.

High Intensity Focused Ultrasound (HIFU) is a very promising technology for a non-invasive treatment of several pathologies, especially in oncology. However, optimizing the stimulation parameters for better tuning the induced lethal effects (thermal and/or mechanical) in the targeted area is not trivial and it has not been achieved yet. The aim of this study is to present the results of a combined analysis of temperature, acoustic cavitation and lesion geometry induced in ex vivo tissues during HIFU procedures by varying power, sonication time and duty cycle. Temperature rise was analyzed using a thin wire thermocouple embedded in the sonicated tissue; stable and inertial cavitation were measured using a passive cavitation detector (PCD), and lesion volume was assessed using both ultrasound imaging and optical visualization. The obtained results may represent an important guideline for clinical treatments, providing useful nformation for better tuning HIFU operational parameters to induce a desired type of ablation (i.e. thermal, mechanical or a combination of both).

Motion compensation with skin contact control for high intensity focused ultrasound surgery in moving organs.

High intensity focused ultrasound (HIFU) is an emerging therapeutic solution that enables non-invasive treatment of several pathologies, mainly in oncology. On the other hand, accurate targeting of moving abdominal organs (e.g. liver, kidney, pancreas) is still an open challenge. This paper proposes a novel method to compensate the physiological respiratory motion of organs during HIFU procedures, by exploiting a robotic platform for ultrasound-guided HIFU surgery provided with a therapeutic annular phased array transducer. The proposed method enables us to keep the same contact point between the transducer and the patient's skin during the whole procedure, thus minimizing the modification of the acoustic window during the breathing phases. The motion of the target point is compensated through the rotation of the transducer around a virtual pivot point, while the focal depth is continuously adjusted thanks to the axial electronically steering capabilities of the HIFU transducer. The feasibility of the angular motion compensation strategy has been demonstrated in a simulated respiratory-induced organ motion environment. Based on the experimental results, the proposed method appears to be significantly accurate (i.e. the maximum compensation error is always under 1 mm), thus paving the way for the potential use of this technique for in vivo treatment of moving organs, and therefore enabling a wide use of HIFU in clinics.

Ultrasound Acoustic Radiation Force Impulse imaging for High Intensity Focused Ultrasound focal spot localization.

Focal spot precise localization highly contributes to the accuracy and safety of High Intensity Focused Ultrasound (HIFU) therapies, and it is usually performed by means of Magnetic Resonance-Acoustic Radiation Force Impulse imaging (MR-ARFI). Acoustic Radiation Force Impulse imaging using ultrasound (US-ARFI) is herein proposed as a valid alternative to MR-ARFI for an accurate and non-destructive detection of the focal spot position during the pre-treatment phase. To this aim, a system composed of a HIFU transducer for generating the acoustic radiation force and a 2D confocal ultrasound probe for measuring the induced micro-displacement have been used. Then, an algorithm based on the Normalized Cross Correlation was implemented for the creation of a displacement map in which the highest displacement area, corresponding to the focal spot region, is unequivocally visualized. The feasibility of the proposed USARFI method for HIFU focal spot localization was successfully demonstrated in a tissue mimicking phantom model.

Asymptomatic spontaneous migration of double pigtail ureteral stent outside the ureter.

We report a case of asymptomatic spontaneous migration outside the ureter of a double pigtail ureteral stent. This previously undescribed complication confirms the need for regular follow-up of patients with indwelling stents.

Tuning acoustic and mechanical properties of materials for ultrasound phantoms and smart substrates for cell cultures.

Materials with tailored acoustic properties are of great interest for both the development of tissue-mimicking phantoms for ultrasound tests and smart scaffolds for ultrasound mediated tissue engineering and regenerative medicine. In this study, we assessed the acoustic properties (speed of sound, acoustic impedance and attenuation coefficient) of three different materials (agarose, polyacrylamide and polydimethylsiloxane) at different concentrations or cross-linking levels and doped with different concentrations of barium titanate ceramic nanoparticles. The selected materials, besides different mechanical features (stiffness from few kPa to 1.6MPa), showed a wide range of acoustic properties (speed of sound from 1022 to 1555m/s, acoustic impedance from 1.02 to 1.67MRayl and attenuation coefficient from 0.2 to 36.5dB/cm), corresponding to ranges in which natural soft tissues can fall. We demonstrated that this knowledge can be used to build tissue-mimicking phantoms for ultrasound-based medical procedures and that the mentioned measurements enable to stimulate cells with a highly controlled ultrasound dose, taking into account the attenuation due to the cell-supporting scaffold. Finally, we were able to correlate for the first time the bioeffect on human fibroblasts, triggered by piezoelectric barium titanate nanoparticles activated by low-intensity pulsed ultrasound, with a precise ultrasound dose delivered. These results may open new avenues for the development of both tissue-mimicking materials for ultrasound phantoms and smart triggerable scaffolds for tissue engineering and regenerative medicine. STATEMENT OF SIGNIFICANCE: This study reports for the first time the results of a systematic acoustic characterization of agarose, polyacrylamide and polydimethylsiloxane at different concentrations and cross-linking extents and doped with different concentrations of barium titanate nanoparticles. These results can be used to build tissue-mimicking phantoms, useful for many ultrasound-based medical procedures, and to fabricate smart materials for stimulating cells with a highly controlled ultrasound dose. Thanks to this knowledge, we correlated for the first time a bioeffect (the proliferation increase) on human fibroblasts, triggered by piezoelectric nanoparticles, with a precise US dose delivered. These results may open new avenues for the development of both tissue-mimicking phantoms and smart triggerable scaffolds for tissue engineering and regenerative medicine.

Speed of sound in rubber-based materials for ultrasonic phantoms.

PURPOSE: In this work we provide measurements of speed of sound (SoS) and acoustic impedance (Z) of some doped/non-doped rubber-based materials dedicated to the development of ultrasound phantoms. These data are expected to be useful for speeding-up the preparation of multi-organ phantoms which show similar echogenicity to real tissues. METHODS: Different silicones (Ecoflex, Dragon-Skin Medium) and polyurethane rubbers with different liquid (glycerol, commercial detergent, N-propanol) and solid (aluminum oxide, graphene, steel, silicon powder) inclusions were prepared. SoS of materials under investigation was measured in an experimental setup and Z was obtained by multiplying the density and the SoS of each material. Finally, an anatomically realistic liver phantom has been fabricated selecting some of the tested materials. RESULTS: SoS and Z evaluation for different rubber materials and formulations are reported. The presence of liquid additives appears to increase the SoS, while solid inclusions generally reduce the SoS. The ultrasound images of realized custom fabricated heterogeneous liver phantom and a real liver show remarkable similarities. CONCLUSIONS: The development of new materials' formulations and the knowledge of acoustic properties, such as speed of sound and acoustic impedance, could improve and speed-up the development of phantoms for simulations of ultrasound medical procedures.

A computer-assisted robotic platform for Focused Ultrasound Surgery: Assessment of high intensity focused ultrasound delivery.

In the last century, medicine showed considerable advancements in terms of new technologies, devices and diagnostic/therapeutic strategies. Those advantages led to a significant reduction of invasiveness and an improvement of surgical outcomes. In this framework, a computer-assisted surgical robotic platform able to perform non-invasive Focused Ultrasound Surgery (FUS) - the FUTURA platform - has the ambitious goal to improve accuracy, safety and flexibility of the treatment, with respect to current FUS procedures. Aim of this work is to present the current implementation of the robotic platform and the preliminary results about high intensity focused ultrasound (HIFU) delivery in in-vitro conditions, under 3D ultrasound identification and monitoring. Tests demonstrated that the average accuracy of the HIFU delivery is lower than 0.7 mm in both X and Y radial directions and 3.7 mm in the axial direction (Z) with respect to the HIFU transducer active surface.

Therapy of Klebsiella pneumoniae septicaemia in neutropenic rats.

The efficacy of intramuscular gentamicin, sisomicin, tobramycin and amikacin was evaluated in the antimicrobial therapy of septicaemia due to Klebsiella pneumoniae in an experimental model of infection in rats rendered neutropenic by cyclophosphamide. Animals were injected with a LD50 of micro-organisms and 4 hours later treated with a therapeutic i.m. dose of the antibiotics. In animals sacrificed at 0.5, 1 and 4 hours after antibiotic treatment, blood levels of aminoglycosides, bactericidal power of serum and quantitative cultures of peritoneal fluid and blood showed that the four antibiotics tested were effective in drastically reducing the number of bacteria in blood and in the peritoneum, concurrently with the bactericidal power of the serum, though with sisomicin preceding in order of activity gentamicin, amikacin and tobramycin.

[Isolation and significance of Vibrio cholera NAG]. / Isolamento e significato di Vibrio cholerae NAG.

After the isolation of two Vibrio cholerae NAG from the stools of two tourists, the authors researched Vibrio in people coming home from particular countries and in resident people. The research was extended to the water of Varese lake after another isolation from a fisher who had fished, cooked and eaten the lake fish. Problems concerning the classification of Vibrio and their presence in the environment are examined.

[Antibacterial activity in vitro of several aminoglycosides and cephalosporins]. / Attivita' antibatterica "in vitro" di alcuni aminoglicosidi e cefalosporine.

The "in vitro" rate of killing on different microorganisms was studied for three cephalosporins (cephazolin, cefuroxime and cefoxitin) and four aminoglycosides (gentamicin, tobramycin, sisomicin and amikacin). In all experiments an inoculum effect and a negative influence by adding plasma to the nutrient broth were observed, the latter phenomenon being more pronounced with cephalosporins than with aminoglycosides. While the "in vitro" effects of antibiotic concentrations equal to the MIC or to the MBC were informing on the intrinsic antibacterial activity of single drugs, a more satisfactory approach to therapeutic reality was provided by studying the "in vitro" effects of concentrations equal to peak blood level or to the logarythmic mean of blood level, that is a parameter which contributes to the blood-tissue diffusion gradient. Our data show that antibiotics provided with an higher peak blood level and a more sustained logarythmic mean of blood concentrations, and an higher ratio between these two kinetics parameters and MIC or MBC, are favoured in the experimental system here adopted.