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.

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 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.

Functional characterization of iPSC-derived arterial- and venous-like endothelial cells.

The current work reports the functional characterization of human induced pluripotent stem cells (iPSCs)- arterial and venous-like endothelial cells (ECs), derived in chemically defined conditions, either in monoculture or seeded in a scaffold with mechanical properties similar to blood vessels. iPSC-derived arterial- and venous-like endothelial cells were obtained in two steps: differentiation of iPSCs into endothelial precursor cells (CD31pos/KDRpos/VE-Cadmed/EphB2neg/COUP-TFneg) followed by their differentiation into arterial and venous-like ECs using a high and low vascular endothelial growth factor (VEGF) concentration. Cells were characterized at gene, protein and functional levels. Functionally, both arterial and venous-like iPSC-derived ECs responded to vasoactive agonists such as thrombin and prostaglandin E2 (PGE2), similar to somatic ECs; however, arterial-like iPSC-derived ECs produced higher nitric oxide (NO) and elongation to shear stress than venous-like iPSC-derived ECs. Both cells adhered, proliferated and prevented platelet activation when seeded in poly(caprolactone) scaffolds. Interestingly, both iPSC-derived ECs cultured in monoculture or in a scaffold showed a different inflammatory profile than somatic ECs. Although both somatic and iPSC-derived ECs responded to tumor necrosis factor-α (TNF-α) by an increase in the expression of intercellular adhesion molecule 1 (ICAM-1), only somatic ECs showed an upregulation in the expression of E-selectin or vascular cell adhesion molecule 1 (VCAM-1).

Influence of substrate stiffness on human induced pluripotent stem cells: preliminary results.

Skeletal muscle differentiation was proven to be influenced by changes in the substrate stiffness. However, a lack of knowledge features this field, concerning skeletal muscle tissues obtained from human induced pluripotent stem cells. Here we report the fabrication of polydimethylsiloxane-based substrates in a range of stiffness values from 3.5 to 141 kPa and the response of human induced pluripotent stem cells cultured on them for 5 days. The substrates were able to sustain cell adhesion and proliferation throughout the whole period. An inversely proportional relationship (although not significant) was found between the proliferation rate and the substrate stiffness. Initial analyses of iPSCs skeletal muscle differentiation shown no influences on markers of the early stages. These results lay the foundations for further studies on the influence of extrinsic mechanical stimuli on induced pluripotent stem cells-derived skeletal muscle tissues.

Nanocomposite thin films based on polyethylene vinyl acetate and piezoelectric nanomaterials.

This paper aims at describing the fabrication and characterization of nanocomposite thin films based on polyethylene vinyl acetate, at different content of vinyl acetate, and piezoelectric nanomaterials, namely zinc oxide and barium titanate. These membranes were prepared by casting, achieving a thickness in the order of 160-210 µm. The nanocomposites were characterized in terms of morphological, mechanical and chemical properties, finding a homogeneous distribution of nanomaterials, and the elastic modulus ranges from 2 to 25 MPa, while keeping an elongation break from 750 to 1500 % and tensile strength from 2.5 MPa up to 10 MPa. These results show the potential of these nanocomposite formulations as smart composite thin films for a series of biomedical applications, including the regeneration of osteoarticular tissues.

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.

Flexible nanofilms coated with aligned piezoelectric microfibers preserve the contractility of cardiomyocytes.

The use of engineered cardiac tissue for high-throughput drug screening/toxicology assessment remains largely unexplored. Here we propose a scaffold that mimics aspects of cardiac extracellular matrix while preserving the contractility of cardiomyocytes. The scaffold is based on a poly(caprolactone) (PCL) nanofilm with magnetic properties (MNF, standing for magnetic nanofilm) coated with a layer of piezoelectric (PIEZO) microfibers of poly(vinylidene fluoride-trifluoroethylene) (MNF+PIEZO). The nanofilm creates a flexible support for cell contraction and the aligned PIEZO microfibers deposited on top of the nanofilm creates conditions for cell alignment and electrical stimulation of the seeded cells. Our results indicate that MNF+PIEZO scaffold promotes rat and human cardiac cell attachment and alignment, maintains the ratio of cell populations overtime, promotes cell-cell communication and metabolic maturation, and preserves cardiomyocyte (CM) contractility for at least 12 days. The engineered cardiac construct showed high toxicity against doxorubicin, a cardiotoxic molecule, and responded to compounds that modulate CM contraction such as epinephrine, propranolol and heptanol.

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.

Polydimethylsiloxane films doped with NdFeB powder: magnetic characterization and potential applications in biomedical engineering and microrobotics.

This work reports the fabrication, magnetic characterization and controlled navigation of film-shaped microrobots consisting of a polydimethylsiloxane-NdFeB powder composite material. The fabrication process relies on spin-coating deposition, powder orientation and permanent magnetization. Films with different powder concentrations (10 %, 30 %, 50 % and 70 % w/w) were fabricated and characterized in terms of magnetic properties and magnetic navigation performances (by exploiting an electromagnet-based platform). Standardized data are provided, thus enabling the exploitation of these composite materials in a wide range of applications, from MEMS/microrobot development to biomedical systems. Finally, the possibility to microfabricate free-standing polymeric structures and the biocompatibility of the proposed composite materials is demonstrated.

Self-assembly of polydimethylsiloxane structures from 2D to 3D for bio-hybrid actuation.

This work aims to demonstrate the feasibility of a novel approach for the development of 3D self-assembled polydimethylsiloxane structures, to be used as engineered flexible matrices for bio-hybrid actuation. We described the fabrication of engineered bilayers, organized in a 3D architecture by means of a stress-induced rolling membrane technique. Such structures were provided with ad hoc surface topographies, for both cell alignment and cell survival after membrane rolling. We reported the results of advanced finite element model simulations, predicting the system behavior in terms of overall contraction, induced by the contractile activity of muscle cells seeded on the membrane. Then, we tested in vitro the structure with primary cardiomyocytes to evaluate the real bio-actuator contraction, thus validating the simulation results. At a later stage, we provided the samples with a stable fibronectin coating, by covalently binding the protein on the polymer surface, thus enabling long-term cultures with C2C12 skeletal muscle cells, a more controllable cell type. These tests revealed cell viability and alignment on the rolled structures, but also the ability of cells to differentiate and to form multinucleated and oriented myotubes on the polymer surface, also supported by a fibroblast feeder layer. Our results highlighted the possibility of developing 3D rolled PDMS structures, characterized by different mechanical properties, as novel bio-hybrid actuators.

Magnetically driven microrobotic system for cancer cell manipulation.

Lab-on-a-chip applications, such as single cell manipulation and targeted delivery of chemicals, could greatly benefit from mobile untethered microdevices able to move in fluidic environments by using magnetic fields. In this paper a magnetically driven microrobotic system enabling the controlled locomotion of objects placed at the air/liquid interface is proposed and exploited for cell manipulation. In particular authors report the design, fabrication and testing of a polymeric thin film-based magnetic microrobot (called "FilmBot") used as a support for navigating cancer cells. By finely controlling magnetic film locomotion, it is possible to navigate the cells by exploiting their adhesion to the film without affecting their integrity. Preliminary in vitro tests demonstrated that the magnetic thin film is able to act as substrate for T24 bladder cancer cells without affecting their viability and that film locomotion can be magnetically controlled (with a magnetic field and a gradient of 6 mT and 0.6 T/m, respectively) along specific directions, with a mean speed of about 3 mm/s.

Investigation of interactions between poly-L-lysine-coated boron nitride nanotubes and C2C12 cells: up-take, cytocompatibility, and differentiation.

Boron nitride nanotubes (BNNTs) have generated considerable interest within the scientific community by virtue of their unique physical properties, which can be exploited in the biomedical field. In the present in vitro study, we investigated the interactions of poly-l-lysine-coated BNNTs with C2C12 cells, as a model of muscle cells, in terms of cytocompatibility and BNNT internalization. The latter was performed using both confocal and transmission electron microscopy. Finally, we investigated myoblast differentiation in the presence of BNNTs, evaluating the protein synthesis of differentiating cells, myotube formation, and expression of some constitutive myoblastic markers, such as MyoD and Cx43, by reverse transcription - polymerase chain reaction and Western blot analysis. We demonstrated that BNNTs are highly internalized by C2C12 cells, with neither adversely affecting C2C12 myoblast viability nor significantly interfering with myotube formation.

Potentiation of antiproliferative drug activity by lonidamine in hepatocellular carcinoma cells.

The ability of lonidamine (LND), a derivative of indazole-carboxylic acid, to modulate the cytotoxic activity of anticancer drugs was investigated in two human hepatocarcinoma (HCC) cell lines. The cytotoxicity of drugs used singly, in association or in sequence was evaluated using the Sulforhodamine B (SRB) assay. LND did not appreciably potentiate the effect of antitumor drugs when given before or simultaneously, in either cell line. Conversely, a synergistic interaction was observed in both cell lines when LND was given after conventional drugs. LND produced a moderate decrease in S-phase cell fraction and did not induce apoptosis. Conversely, paclitaxel (TAX) induced an important block in G2 and an increase in apoptosis. Following a 48-h TAX wash out, a progressive passage of cells from G2 to M phase was observed with a corresponding increase in apoptotic cells. Post-treatment with LND increased the cytotoxicity of some antitumor drugs, especially TAX, in hepatocarcinoma cells, possibly by preventing, as an energolytic drug, cell damage repair or by producing an additional effect on microtubule stabilization.

Doxorubicin, paclitaxel and gemcitabine: a Phase I study of a new sequential treatment in stage III B - IV breast cancer.

Based on the synergistic interactions of the sequence doxorubicin-paclitaxel-gemcitabine obtained in our preclinical study, a Phase I trial was conducted to evaluate the feasibility of this new sequence in breast cancer. Patients with stage IIIB-IV breast cancer received doxorubicin on day 1, paclitaxel on day 2 and gemcitabine on day 6 and 13 (steps IIa, III and V) in cohorts of 3 patients. From March 1999 to December 2000, 9 patients were treated. The most important toxicity was hematological. The maximum tolerated dose was reached at the second level because dose-limiting toxicity occurred in 3 patients. Non hematological toxicities were alopecia, diarrhea, asthenia, nausea, mucositis, paresthesia and myalgia. A Phase II trial is ongoing to further investigate the activity of this new sequential treatment with doxorubicin (50 mg/m2 day 1), paclitaxel (160 mg/m2 day 2) and gemcitabine (800 mg/m2 day 6) in advanced breast cancer.

Flow-cytometric determination of tumor cells in lymph nodes.

In solid tumors, metastasis occurs through the dissemination of tumor cells in the bloodstream and the lymphatic system. In particular, lymph node infiltration gives useful prognostic information and represents one of the most important factors for selecting the type of clinical treatment in disease management. Furthermore, the analysis of lymph node infiltration has become important for identifying patients with breast cancer or malignant melanoma who may be candidates for regional lymph node dissection. Tumor cells in lymph nodes are currently identified in tissue sections using morphological and immunohistochemical analyses, but these approaches are time-consuming, and micrometastases may escape detection. The aim of the present study was to define the potential of a flow cytometric (FCM) determination based on cell size and autofluorescence to shorten the time required for lymph node analysis. The sensitivity of the FCM approach, defined on mixtures of tumor cells from established cell lines and peripheral blood lymphocytes (PBL(s)) at different concentrations, was 1 tumor cell/1,000 PBL(s). FCM analysis was performed on 89 lymph nodes, 29 from breast, 41 from lung and 19 from colon cancer patients. Agreement between FCM and morphological results, used as gold standard, was observed in 83% of the cases, and there was a 90% sensitivity to the FCM approach for each tumor type. Disagreement was observed for 15 lymph nodes and was due, in the majority of cases (80%), to FCM-positive and morphologically negative results. A large number of patients and a more accurate pathological examination of consecutive histological sections of lymph nodes are needed to further evaluate the validity of the FCM approach.

In vitro preclinical models for a rational design of chemotherapy combinations in human tumors.

Today, drug combinations are frequently used in the treatment of cancer to increase therapeutic efficacy. Currently used clinical protocols for cancer combination therapies are mainly obtained empirically or on the basis of results from previous clinical trials. Information obtained from clinical protocols is invaluable, but it is time-consuming, expensive and does not provide data on the biochemical and molecular mechanisms of interaction of the drugs used in combination treatments at cellular level. Therefore, in vitro drug combination studies on established cell lines or primary cell cultures play an important role in designing and optimising combination protocols. A variety of in vitro assays and different mathematics models have been developed to investigate cytotoxic effects and to analyse the type of drug interactions. Increased knowledge of the cellular targets of traditional and new drugs and the development of new technologies have resulted in a new role for the in vitro tests which are no longer used only to evaluate the cytotoxic effects of drugs, but also to investigate the interference on cell cycle, induction of apoptosis and molecular or biochemical interactions. A review on in vitro preclinical tests used to evaluate the effects of drug combinations and to design the rationale of combined chemotherapy protocols is presented.

Simultaneous determination of apoptosis and surface antigen expression in tumor adherent cells.

Apoptosis is a physiological, gene-directed form of cell death aimed at controlling cell proliferation in several biological conditions. It plays a crucial role in modulating tissue growth during embryonic development, cell turnover in adult life, and it seems to be the most frequent mechanism of tumor cell deletion by chemotherapy. Flow cytometry is a widely-used technique for checking apoptosis, permitting a multiparametric analysis. It is possible to follow the alterations occurring in the nucleus, mitochondria and plasmatic membrane during the different apoptotic stages using probes such as LDS-751, JC-1 or Annexin V. The potential of these probes to identify the early or late stages of apoptosis has been widely investigated in cells growing in suspension. In order to assess apoptosis in adherent cells, we tested a combination of fluorescein diacetate (FDA), a substrate for non specific esterase whose activity decreases during the early phase of apoptosis, and trypan blue in MCF-7 human breast cancer cells. Apoptotic cells showed a decrease in the green fluorescence emitted by fluorescein, the product of FDA hydrolysis, whereas necrotic cells emitted a red fluorescence due to the trypan blue staining. FDA-trypan blue double-staining was used to investigate the different kinetics of apoptosis induced by taxol, camptothecin and UV-B irradiation in MCF-7 cells. This method is rapid and simple, and can be used for monitoring the process of apoptosis from early stages in adherent cells, for the physical separation of apoptotic and live cells, and for immunophenotyping, including Fas expression.

Lanreotide-induced modulation of 5-fluorouracil or mitomycin C cytotoxicity in human colon cancer cell lines: a preclinical study.

The effect on growth of the long-acting somatostatin analogue lanreotide (LAN), alone or in combination with 5-fluorouracil (5-FU) and mitomycin C (MIT), was investigated in three human colon cancer lines. Cell survival inhibition induced by LAN alone, as evaluated by sulforhodamine B assay, ranged from 20% to 40% as a function of cell line and concentration. The IC50, the concentration inhibiting cell survival by 50%, was never reached. The antiproliferative effect produced by a 48 h exposure to 5-FU or MIT was synergistically enhanced in all cell lines by a subsequent 48 h exposure to LAN. The synergistic interaction was not related to specific cell cycle perturbations or to the somatostatin receptor 2 (sst2) mRNA abundance. In conclusion, our study seems to indicate that LAN is a potentially useful modulating agent for enhancing 5-FU and MIT activity in colorectal cancer patients.