Comparison between different cell sources and culture strategies for tendon tissue engineering.

The aim of this study was to evaluate the effect of an in vitro mechanical stimulation by the use of a bioreactor on an engineered tendon for 7 and 14 days and to analyze the effect of the use of different cell sources: tenocytes, dermal fibroblasts or Adipose-Derived Stem Cells (ASCs), isolated from pig tissues. Histology showed a re-organization of the neo-tissue derived from the three cell populations along the direction of the stimulus. At T7, cells morphology was preserved while an increased cellular suffering at T14 was observed for all cell populations. Tenocytes exhibited higher survival than other cells. A stable immunopositivity for collagen type 1 or 3 at both time points was also observed. In conclusion, dermal fibroblasts and ASCs represent an interesting alternative and in vitro culture with mechanical stimuli may enhance the maturation of a tendon-like tissue.

No evidence of cardiomyopathy in spinal and bulbar muscular atrophy.

OBJECTIVES: Spinal and bulbar muscular atrophy (SBMA) is a lower motor neuron disease caused by a CAG repeat expansion within the androgen receptor (AR) gene. Toxic nuclear accumulation of mutant AR has been observed in tissues other than nervous system including cardiac muscle. Moreover, CAG polymorphism length within AR has been associated with an increased risk of heart disease. MATERIALS AND METHODS: To test the hypothesis of the presence of cardiomyopathy in SBMA, a full cardiac protocol was applied to 25 SBMA patients. RESULTS: Patients' age ranged between 32 and 75 years. Cardiologic examination, 12-lead ECG, and echocardiography showed no abnormalities other than those consistent with hypertensive heart disease. One patient showed frequent supraventricular premature beats in absence of other significant arrhythmias at the 24-h ECG Holter. CONCLUSIONS: Our findings do not support the hypothesis of a primary cardiomyopathy in SBMA.

Preventing weight gain during adjuvant chemotherapy for breast cancer: a dietary intervention study.

Adjuvant chemotherapy significantly decreases recurrences and improves survival in women with early breast cancer (BC). However, the side effects of chemotherapy include weight gain, which is associated with poorer prognosis. We have previously demonstrated that by means of a comprehensive dietary modification which aims at lowering insulin levels it is possible to reduce body weight and decrease the bioavailability of insulin, sex hormones and the growth factors linked to BC risk and prognosis. We are now going to present a randomized controlled study of adjuvant diet in BC patients undergoing chemotherapy. The diet was designed to prevent weight gain during chemotherapy treatment. Women of any age, operated for invasive BC, scheduled for adjuvant chemotherapy and without evidence of distant metastases, were randomized into a dietary intervention group and a control group. The intervention implied changing their usual diet for the whole duration of chemotherapy, following cooking classes and having lunch or dinner at the study centre at least twice per week. 96 BC patients were included in the study. The women in the intervention group showed a significant reduction in their body weight (2.9 kg on average), compared with the controls. They also significantly reduced body fat mass, waist and hip circumferences, biceps, underscapular and suprailiac skinfolds, compared with the women in the control group. Our results support the hypothesis that dietary intervention during adjuvant chemotherapy for BC is feasible and may prevent weight gain.

Phototoxicity induced in living HeLa cells by focused femtosecond laser pulses: a data-driven approach.

Nonlinear optical microscopy is a powerful label-free imaging technology, providing biochemical and structural information in living cells and tissues. A possible drawback is photodamage induced by high-power ultrashort laser pulses. Here we present an experimental study on thousands of HeLa cells, to characterize the damage induced by focused femtosecond near-infrared laser pulses as a function of laser power, scanning speed and exposure time, in both wide-field and point-scanning illumination configurations. Our data-driven approach offers an interpretation of the underlying damage mechanisms and provides a predictive model that estimates its probability and extension and a safety limit for the working conditions in nonlinear optical microscopy. In particular, we demonstrate that cells can withstand high temperatures for a short amount of time, while they die if exposed for longer times to mild temperatures. It is thus better to illuminate the samples with high irradiances: thanks to the nonlinear imaging mechanism, much stronger signals will be generated, enabling fast imaging and thus avoiding sample photodamage.

Molecular MR imaging for the evaluation of the effect of dynamic stabilization on lumbar intervertebral discs.

The dynamic stabilization of lumbar spine is a non-fusion stabilization system that unloads the disc without the complete loss of motion at the treated motion segment. Clinical outcomes are promising but still not definitive, and the long-term effect on instrumented and adjacent levels is still a matter of discussion. Several experiments have been devised in order to gain a better understanding of the effect of the device on the intervertebral disc. One of the hypotheses was that while instrumented levels are partially relieved from loading, adjacent levels suffer from the increased stress. But this has not been proved yet. The aim of this study was to investigate the long-term effect of dynamic stabilization in vivo, through the quantification of glycosaminoglycans (GAG) concentration within instrumented and adjacent levels by means of the delayed Gadolinium-Enhanced Magnetic Resonance Imaging of Cartilage (dGEMRIC) protocol. Ten patients with low back pain, unresponsive to conservative treatment and scheduled for Dynesys implantation at one to three lumbar spine levels, underwent the dGEMRIC protocol to quantify GAG concentration before and 6 months after surgery. Each patient was also evaluated with visual analog scale (VAS), Oswestry, Prolo, Modic and Pfirrmann scales, both at pre-surgery and at follow-up. Six months after implantation, VAS, Prolo and Oswestry scales had improved in all patients. Pfirrmann scale could not detect any change, while dGEMRIC data already showed a general improvement in the instrumented levels: GAG was increased in 61% of the instrumented levels, while 68% of the non-instrumented levels showed a decrease in GAG, mainly in the posterior disc portion. In particular, seriously GAG-depleted discs seemed to have the greatest benefit from the Dynesys implantation, whereas less degenerated discs underwent a GAG depletion. dGEMRIC was able to visualize changes in both instrumented and non-instrumented levels. Our results suggest that the dynamic stabilization of lumbar spine is able to stop and partially reverse the disc degeneration, especially in seriously degenerated discs, while incrementing the stress on the adjacent levels, where it induces a matrix suffering and an early degeneration.

Mechanosensing at the Nuclear Envelope by Nuclear Pore Complex Stretch Activation and Its Effect in Physiology and Pathology.

Cell fate is correlated to mechanotransduction, in which forces transmitted by the cytoskeleton filaments alter the nuclear shape, affecting transcription factor import/export, cells transcription activity and chromatin distribution. There is in fact evidence that stem cells cultured in 3D environments mimicking the native niche are able to maintain their stemness or modulate their cellular function. However, the molecular and biophysical mechanisms underlying cellular mechanosensing are still largely unclear. The propagation of mechanical stimuli via a direct pathway from cell membrane integrins to SUN proteins residing in the nuclear envelop has been demonstrated, but we suggest that the cells' fate is mainly affected by the force distribution at the nuclear envelope level, where the SUN protein transmits the stimuli via its mechanical connection to several cell structures such as chromatin, lamina and the nuclear pore complex (NPC). In this review, we analyze the NPC structure and organization, which have not as yet been fully investigated, and its plausible involvement in cell fate. NPC is a multiprotein complex that spans the nuclear envelope, and is involved in several key cellular processes such as bidirectional nucleocytoplasmic exchange, cell cycle regulation, kinetochore organization, and regulation of gene expression. As several connections between the NPC and the nuclear envelope, chromatin and other transmembrane proteins have been identified, it is reasonable to suppose that nuclear deformations can alter the NPC structure. We provide evidence that the transmission of mechanical forces may significantly affects the basket conformation via the Nup153-SUN1 connection, both altering the passage of molecules through it and influencing the state of chromatin packing. Finally, we review the known correlations between a pathological NPC structure and diseases such as cancer, autoimmune disease, aging and laminopathies.

Chondrocyte response to high regimens of cyclic hydrostatic pressure in 3-dimensional engineered constructs.

PURPOSE: Despite widespread use of 3-dimensional (3D) micro-porous scaffolds to promote their potential application in cartilage tissue engineering, only a few studies have examined the response to hydrostatic pressure of engineered constructs. A high cyclic pressurization, currently believed to be the predominant mechanical signal perceived by cells in articular cartilage, was used here to stimulate bovine articular chondrocytes cultured in a synthetic 3D porous scaffold (DegraPol). METHODS: Construct cultivation lasted 3 days with applied pressurization cycles of amplitude 10 MPa, frequency 0.33 Hz, and stimulation sessions of 4 hours/day. RESULTS: At 3 days of culture, with respect to pre-culture conditions, the viability of the pressurized constructs did not vary, whereas it underwent a 16% drop in the unpressurized controls. Synthesis of alfa-actin was 34% lower in all cultured constructs. Synthesis of collagen II/collagen I did not vary in pressurized constructs, was 76% lower in unpressurized controls, and was around 230% higher in pressurized constructs with respect to unpressurized controls. Chondrocytes showed a phenotypic spherical morphology at time zero and at 3 days of pressurized culture. CONCLUSIONS: Although the passage from 2D expansion to 3D geometry was effective to guide cell differentiation, only mechanical conditioning enabled the maintenance and further cell differentiation toward a mature chondrocytic phenotype.

A new bioreactor for the controlled application of complex mechanical stimuli for cartilage tissue engineering.

Mechanical stimuli have been shown to enhance chondrogenesis on both animal and human chondrocytes cultured in vitro. Different mechanical stimuli act simultaneously in vivo in cartilage tissue and their effects have been extensively studied in vitro, although often in a separated manner. A new bioreactor is described where different mechanical stimuli, i.e. shear stress and hydrostatic pressure, can be combined in different ways to study the mechanobiology of tissue engineered cartilage. Shear stress is imposed on cells by forcing the culture medium through the scaffolds, whereas a high hydrostatic pressure up to 15 MPa is generated by pressurizing the culture medium. Fluid-dynamic experimental tests have been performed and successful validation of the bioreactor has been carried out by dynamic culture of tissue-engineered cartilage constructs. The bioreactor system allows the investigation of the combined effects of different mechanical stimuli on the development of engineered cartilage, as well as other possible three-dimensional tissue-engineered constructs.

Parametric FE mesh generation: application to the cervical spine.

A voxel-based reconstruction algorithm, targeted at the generation of finite element (FE) meshes of structures with schematic geometry, is presented. The algorithm is able to generate three dimensional fully hexahedral FE meshes of structures composed of volumes with a schematic geometry. In order to be meshed, each volume must be described in terms of a set of surfaces which enclose the volume. Due to its schematic nature, the method allows the generation of fully parameterized FE models, thus it facilitates the investigation of the mechanical relevance of geometrical parameters by speeding up the mesh generation process. The algorithm was employed in the automatic construction of an FE model of the C3-C7 spinal segment with schematic geometry, made up exclusively of hexahedral elements. Non-linear simulations were carried out in different loading conditions: flexion- extension, lateral bending and axial rotation. The results were compared to data retrieved from the literature in order to ensure the validity of the model. Moment-rotation curves for each loading condition were determined. The range of motion was obtained for each spinal unit and loading condition. Both principal and coupled rotations were determined in lateral bending and axial rotation, for each spinal unit. The intradiscal pressure was also computed in the nucleus pulposus, for all the intervertebral levels. Geometrical parameterization of the models offers potential for the biomechanical investigation of pathologic conditions and surgical procedures, such as spinal fusion and disc arthroplasty, even on a patient-specific basis.

Biomechanical regulation of drug sensitivity in an engineered model of human tumor.

Predictive testing of anticancer drugs remains a challenge. Bioengineered systems, designed to mimic key aspects of the human tumor microenvironment, are now improving our understanding of cancer biology and facilitating clinical translation. We show that mechanical signals have major effects on cancer drug sensitivity, using a bioengineered model of human bone sarcoma. Ewing sarcoma (ES) cells were studied within a three-dimensional (3D) matrix in a bioreactor providing mechanical loadings. Mimicking bone-like mechanical signals within the 3D model, we rescued the ERK1/2-RUNX2 signaling pathways leading to drug resistance. By culturing patient-derived tumor cells in the model, we confirmed the effects of mechanical signals on cancer cell survival and drug sensitivity. Analyzing human microarray datasets, we showed that RUNX2 expression is linked to poor survival in ES patients. Mechanical loadings that activated signal transduction pathways promoted drug resistance, stressing the importance of introducing mechanobiological cues into preclinical tumor models for drug screening.

Computational modeling of combined cell population dynamics and oxygen transport in engineered tissue subject to interstitial perfusion.

This work presents a computational model of tissue growth under interstitial perfusion inside a tissue engineering bioreactor. The model accounts both for the cell population dynamics, using a model based on cellular automata, and for the hydrodynamic microenvironment imposed by the bioreactor, using a model based on the Lattice-Boltzmann equation and the convection-diffusion equation. The conditions of static culture versus perfused culture were compared, by including the population dynamics along with oxygen diffusion, convective transport and consumption. The model is able to deal with arbitrary complex geometries of the spatial domain; in the present work, the domain modeled was the void space of a porous scaffold for tissue-engineered cartilage. The cell population dynamics algorithm provided results which qualitatively resembled population dynamics patterns observed in experimental studies, and these results were in good quantitative agreement with previous computational studies. Simulation of oxygen transport and consumption showed the fundamental contribution of convective transport in maintaining a high level of oxygen concentration in the whole spatial domain of the scaffold. The model was designed with the aim to be computationally efficient and easily expandable, i.e. to allow straightforward implementability of further models of complex biological phenomena of increasing scientific interest in tissue engineering, such as chemotaxis, extracellular matrix deposition and effect of mechanical stimulation.

Biomechanics of the C5-C6 spinal unit before and after placement of a disc prosthesis.

The study consists of a biomechanical comparison between the intact C5-C6 spinal segment and the same segment implanted with the Bryan artificial disc prosthesis (Medtronic Ltd., Memphis, TN, USA), by the use of the finite element (FE) method. Our target is the prediction of the influence of prosthesis placement on the resulting mechanics of the C5-C6 spine unit. A FE model of the intact C5-C6 segment was built, employing realistic models of the vertebrae, disc and ligaments. Simulations were conducted imposing a compression preload combined to a flexion/extension moment, a pure lateral bending moment and a pure torsion moment, and the calculated results were compared to data from literature. The model was then modified to include the Bryan cervical disc prosthesis, and the simulations were repeated. The location of the instantaneous center of rotation (ICR) of C5 with respect to C6 throughout flexion/extension was calculated in both models. In general, the moment-rotation curves obtained from the disc prosthesis-implanted model were comparable to the curves obtained from the intact model, except for a slightly greater stiffness induced by the artificial disc. The position of the calculated ICRs was rather stable throughout flexion-extension and was generally confined to a small area, qualitatively matching the corresponding physiological region, in both models. These results imply that the Bryan disc prosthesis allows to correctly reproduce a physiological flexion/extension at the implanted level. The results of this study have quantified aspects that may assist in optimizing cervical disc replacement primarily from a biomechanical point of view.

[Determinants of depression in 111 Italian patients with systemic sclerosis]. / Determinanti della depressione in 111 pazienti italiani con sclerosi sistemica.

BACKGROUND: A high prevalence of depressive symptoms has been described in systemic sclerosis (SSc), but no clear association with organ involvement or objective indices of disease severity has been depicted. To date, no effort has been made to determine the prevalence of depressive symptoms in Italian patients with SSc or to clarify their cause. METHODS: One-hundred-eleven SSc patients were asked to fill in the Beck Depression Inventory (BDI) questionnaire, the scleroderma Health Assessment Questionnaire (sHAQ) and two additional questions assessing the patient's familiar support and the social consequences of the patient's change in physical appearnace. RESULTS: Thirty-seven subjects (33.4%) presented mild to severe depressive symptoms (BDI >/=17). On univariate analysis the diffuse cutaneous form of the disease (p=0.019), higher pulmonary systolic pressures on echocardiogram (p=0.016), lower FVC percentage of predicted values (p=0.022), higher sHAQ values (p<0.001) or higher VAS values for pain (p=0.007), lung involvement (p=0.02), Raynaud's phenomenon severity (p=0.002), ulcers severity (p=0.006) or disease severity (p<0.001), were associated with the presence of pathologic depressive symptoms. On multivariate analysis only the VAS for disease severity relevant to BDI scores (p=0.016). Social behaviour changes due to SSc-related physical involvement were reported in 14 patients (38%) with depressive symptoms (p=0,006) and were more likely to be observed in younger patients (p=0.001) with a more severe Raynauds's phenomenon (p=0.013). CONCLUSIONS: Mild to severe depressive symptoms are common in SSc patients especially in those with a worse perception of disease severity, these patients should be carefully monitored and a psychological assistance counselled whenever necessary.

Cell-based biosensors: current trends of the development.

In the last decade, a number of laboratories have developed devices that combine electronic components with living cells, including neurons. These devices can be used as cell-based biosensors or labs-on-a-chip for testing of the tumor cell sensitivity to anti-cancer drugs, detection of toxins and chemical substances and pre-clinical evaluation of new drugs. Here we review briefly the existing types of the cell-based biosensors and the strategies employed to improve these complex devices. We argue that, for the neuron-based biosensors, introduction of structure in the connections of the synaptic network should significantly improve the utility of such devices.

Multibody modeling of the cervical spine in the simulation of flexion-extension after disc arthroplasty.

This paper presents a three-dimensional (3D) multibody model of the cervical spine implanted with an artificial disc. The model was used to predict prosthesis placement influence on the resulting cervical kinematics in a series of patients. The vertebral tract modeled was the C2-C7, and the vertebral geometries were reconstructed from computed tomography (CT) images. The model was used to simulate the flexion-extension motion of the cervical spine in 10 patients implanted with the Prestige commercial disc prosthesis at a single level. For each patient, a geometrical model of the prosthesis was scaled and included in the multibody model to match the size and positioning of the actual prosthesis, as assessed on post-operative radiographs. Simulations of complete flexion-extension were carried out for each patient, and the main parameters relevant to the motion of the vertebral bodies were calculated and compared to data measured from dynamic post-operative radiographs. At the implanted level, the simulated ranges of motion generally agreed with the measured ones, with an average deviation <2 degrees. In addition, the simulated relative angles between vertebral bodies agreed with the measured ones, with minor average differ-ences of 1.2, 1.8 and 2.1 degrees in full flexion, neutral alignment and full extension, respectively. The cervical kinematics after prosthesis placement was influenced both by the design of the artificial joint and by surgical positioning. Therefore, the model presented can be used both to support pre-operative planning for disc arthroplasty and in the optimization of new prostheses design.

[Carpal tunnel syndrome among cashiers in commercial businesses]. / La sindrome del tunnel carpale in cassiere di aziende commerciali.

The Check-out assistants represent a working sector at risk of Work Related Musculo Skeletal Disorders (WMSDs). The aim of our study is to evaluate carpal tunnel syndrome's incidence in check out assistants. Our search was carried out on a sample of 695 female check-out assistants,after a specific risk's valutazion witch "chec-list application", during health surveillance. Subjects with an pand's painful symptoms or paraesthesias along territory of median were submitted to tests thath revial compression of median at wrist: Phalen and Tinel tests. Who were positive to painful symptoms or paraesthesias in both tests, were subjected to electromyographia (E.M.G.), gold standard for carpal tunnel Syndrome diagnosis. This sample was compared with a control group no-exposed to specific risk (all teachers) of equal number, age, sex and working age of our sample. Our results point out that: our study sample show more symptoms and carpal tunnel Syndrome than the control group; tere aren't statistically meaningful differences between test's positive subjects amd EMG positive subjects. We demonstrated that a careful anamnesis and objective exam can replace EMG.

Calpain restrains the stem cells compartment in breast cancer.

CAPNS1 is essential for the stability and function of ubiquitous CAPN1 and CAPN2. Calpain modulates by proteolytic cleavage many cellular substrates and its activity is often deregulated in cancer cells, therefore calpain inhibition has been proposed as a therapeutical strategy for a number of malignancies. Here we show that CAPNS1 depletion is coupled to impairment of MCF7 and MCF10AT cell lines growth on plate and defective architecture of mammary acini derived from MCF10A cells. In soft agar CAPNS1 depletion leads to cell growth increase in MCF7, and decrease in MCF10AT cells. In both MCF7 and MCF10AT, CAPNS1 depletion leads to the enlargement of the stem cell compartment, as demonstrated by mammosphere formation assays and evaluation of stem cell markers by means of FACS and western blot analysis. Accordingly, activation of calpain by thapsigargin treatment leads to a decrease in the stem cell reservoir. The expansion of the cancer stem cell population in CAPNS1 depleted cells is coupled to a defective shift from symmetric to asymmetric division during mammosphere growth coupled to a decrease in NUMB protein level.

Absence of lipid gel-phase domains in seven mammalian cell lines and in four primary cell types.

Fluorescence properties of 6-lauroyl-2-dimethylaminonaphthalene (Laurdan) are used to explore gel and liquid-crystalline phase domains coexistence in membranes of various cell types and in erythrocyte ghosts. Experiments and simulations were performed using liposomes composed of equimolar gel and liquid-crystalline phases in the absence and in the presence of 30 mol% cholesterol. In this model system two distinct coexisting phases can be easily recognized in the absence of cholesterol. When cholesterol is added to this phospholipid mixture, Laurdan parameters characteristic of the gel and of the liquid-crystalline phase are no longer resolvable. Coexisting domains of gel and liquid-crystalline phase were not detected in any of the examined cell membranes as judged by Laurdan excitation and emission Generalized Polarization (GP) spectra. Both in liposomes and in cell membranes, the behaviour of GP values as a function of excitation and emission wavelength corresponds to a homogeneous liquid-crystalline phase, despite the absolute GP values being relatively high, closer to the values observed in gel phase phospholipids. The presence of cholesterol appears to be the major cause for the homogeneity of phospholipids' dynamical properties in natural membranes, properties that appear close to the liquid-ordered phase state, defined to describe model systems with cholesterol concentration > or = 30 mol%.

Integrating live cells with semiconductor devices: A biocompatibility assay.

There is increasing interest in the development of small hybrid cell-semiconductor systems for the non-invasive evaluation of the physiological state of a cell population. These miniature devices can be used in many areas of biomedical applications, ranging from basic research to drug screening during cancer chemosensitivity testing in clinics. A prerequisite for the biological and medical application of these devices is that cells retain their functional and growth properties when in contact with the semiconductor sensor material. The sensor surface is usually coated with dielectric silicon dioxide (SiO2 ) or a silicon nitride layer (Si3 N4 ); therefore, cellular adhesion to these materials and cellular viability on these surfaces are of crucial im-portance. This is especially true for bone cells that are sensitive to the surface microstructure. Therefore, we investigated the short-term (1-7 days) behavior of model bone cells (MG63 human osteosarcoma cells) grown on silicon samples coated with SiO2 . Cell adhesion and morphology were evaluated by scanning electron microscopy (SEM) 1 day after seeding and cell pro-liferation was evaluated by Alamar Blue assay at 2, 3 and 7 days after seeding. No adverse cellular reactions could be detected with these assays suggesting that the tested substrate is suitable for the hybrid cell-semiconductor systems that test bone tumor chemosensitivity.

Cholesterol protects the phospholipid bilayer from oxidative damage.

The measurement of fluorescence lifetime distribution of 1,6-diphenyl-1,3,5-hexatriene is used for the detection of oxidative damage produced in phospholipid membranes by ionizing radiation. The recently developed method is based on the linear relationship between the width of the probe lifetime distribution and the logarithm of the dose. The molecular origin of the damage resides in the production of hydroperoxide residues at the level of acyl chains double bonds. A chemiluminescence assay was used to quantitate the amount of produced hydroperoxides. Consequences of the produced damages include an increased disorder in the upper portion of the bilayer, accompanied by the penetration of water molecules. In the presence of the physiological concentration of cholesterol in phopholipid bilayers, the amount of hydroperoxides produced by ionizing radiation is dramatically reduced. The packing effect of cholesterol in phopholipid bilayers is well recognized, as well as its influence on the reduction of water concentration in the bilayer. The dramatic reduction of hydroperoxides concentration observed when irradiation is performed in the presence of cholesterol probably originates from a steric hindrance to the radical chain reaction through the unsaturated lipids due to the presence of cholesterol.