F. De Terlizzi

Identification of in vitro electropermeabilization equivalent pulse protocols.

Exposure of cells to an external sufficiently strong electric field results in the formation of pores across the membrane. This phenomenon, termed electropermeabilization, permits the transport of poorly permeant molecules into cytosol. In clinical practice, cell membrane permeabilization for drug electrotransfer is achieved using the ESOPE pulse protocol (1000 V/cm, 8 pulses, 100 μs, 5 kHz). The aim of this study was to investigate several combinations of electric field amplitude and pulse number able to induce electropermeabilization as the one observed when the ESOPE protocol was applied. Decreasing electric field amplitudes (1000 to 300 V/cm) in combination with increasing number of pulses (8 to 320) were applied to in vitro MG63 cells. Propidium iodide and Calcein blue AM uptake were used to evaluate cell electropermeabilization and viability. Results showed that the threshold of local electric field needed to obtain electropermeabilization decreased exponentially with increasing the number of pulses delivered (r2 50.92, p < 0.0001). The absorbed dose threshold was dependent on the number of pulses for each voltage applied (r2 50.96, p < 0.0001). In conclusion, the possibility of applying an increased number of pulses rather than increasing the electric field amplitude to perform electropermeabilization, may become an important tool for electropermeabilization - related clinical applications.

Phalangeal ultrasonography in forearm fracture discrimination

Over the last decade, the use of ultrasounds has been developed into an effective tool for investigating bone tissue and predicting the risk of fracture in osteoporosis. Studies have focused on hip and vertebral fractures while no information is available on the use of phalangeal ultrasonography to identify patients with forearm fractures. Thus, the current authors decided to compare 50 postmenopausal women with low energy forearm fractures (Fractured Group) with a control age-matched group of 94 women (Control Group). Measurements were taken at the distal metaphysis of the proximal phalanxes of the hand of the non-fractured arm using the DBM Sonic Bone Profiler. The reproducibility of the method was assessed by amplitude-dependent speed of sound (AD-SoS) CV% = 0.64 and by Ultrasound Bone Profiler Index (UBPI) CV% = 2.38. In the Control Group, the AD-SoS and UBPI mean values and standard deviations were significantly higher compared to the group with fractures (P < 0.0005). The receiver operating characteristic (ROC) curves were calculated and the areas under the curve (AUC) were 0.78 +/- 0.04 for AD-SoS and 0.77 +/- 0.05 for UBPI, respectively. Logistic regression analysis adjusted to age revealed that both AD-SoS (78.2%, ORAD-SoS = 12.03, P < 0.0005) and UBPI (76.0%, ORAD-SoS = 7.39, P < 0.0005) parameters discriminated correctly between fractured and non-fractured control women whereas the association of both parameters could not allow better discrimination. The present results showed that ultrasound investigation at the phalanxes is reproducible and efficiently discriminates between subjects with forearm fractures and those in the control subjects.

Influence of density, elasticity, and structure on ultrasound transmission through trabecular bone cylinders

The aim of this in vitro study is to evaluate the potentiality of quantitative ultrasound (QUS) to separate information on density, elasticity, and structure on specimens of trabecular bone. Fifteen cylinders of spongy bone extracted from equine vertebrae were progressively demineralized and subjected to QUS, micro computed tomography (muCT), Dual energy X-ray absorptiometry (DXA) at various mineralization levels. Eventually all cylinders underwent a compression test to calculate the Youngs modulus. Correlation analysis shows that speed of sound (SOS) is strictly associated to bone mineral density (BMD), Youngs modulus, and all muCT parameters except for degree of anisotropy (DA). Fast wave amplitude (FWA) is directly correlated with bone surface and total volume ratio (BS/TV) and trabecular separation (Tb Sp), and inversely correlated with trabecular number (Tb N). Because muCT parameters were strictly correlated to BMD and Youngs modulus data, partial correlation analysis was performed between SOS, FWA, and structural and elastic data in order to eliminate the effect of density. SOS was significantly correlated to bone volume and total volume ratio (BV/TV), BS/TV, and Youngs modulus, and FWA was significantly correlated to Tb Sp only. These results show that SOS is strongly influenced by volumetric mineral bone density and elastic modulus of the specimen, and FWA is mainly affected by trabecular separation independently on density. Therefore, SOS and FWA are able to provide different and complementary information, at least on trabecular bone samples.

Discriminant capacity of quantitative ultrasound versus dual X-Ray absorptiometry to determine cancellous bone loss in ovariectomized rats

The capacity of dual x-ray absorptiometry and quantitative ultrasound to discriminate bone loss and to predict the mechanical and microarchitectural properties of cancellous bone in an animal model of osteopenia was evaluated. Thirty-five female Sprague-Dawley rats (10 months old) were randomized into three groups: baseline group, 10 rats killed at the beginning of the study; ovx group, 15 rats ovariectomized; and sham group, 10 rats sham operated. At the beginning and end of the study, all the animals underwent osteosonography to record the proximal tail (C3 vertebra) bone speed of sound. Sixteen weeks after surgery, the animals were euthanized and the L5-6 lumbar vertebrae of each rat were excised for densitometric, biomechanical (compression test), and histomorphometric studies. Significant differences were found among the groups for final speed of sound (p = 0.01). The L5 bone mineral density of the ovx group decreased by 12.1% (p = 0.049) and 12.6% (p = 0.035) compared, respectively, with baseline and sham groups. The biomechanical parameters of the ovx group decreased by 15-47% compared with the other groups, showing significant differences between the ovx and sham groups both for maximal stress (p = 0.026) and elastic modulus (p = 0.013). Histomorphometric parameters of the ovx group showed significant decreases in comparison with other groups. Logistic regression analysis showed that dual X-ray absorptiometry and quantitative ultrasound discriminate ovariectomized and healthy rats with a similar capacity, classifying correctly all rats used in the model in a range of 61-70%. This similar capacity seems to derive from two different capacities to detect bone changes. Dual X-ray absorptiometry, depending on bone mineralization and density, is able to detect modifications in bone stiffness and strength, confirmed also by the correlation with biomechanical data. On the contrary, quantitative ultrasound seems to depend more on cancellous bone microarchitecural changes because it is correlated to histomorphometric parameters.