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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

A comparative evaluation of chondrocyte/scaffold constructs for cartilage tissue engineering.

This study aimed to evaluate three biodegradable scaffolds as cell carriers for in vitro cartilage regeneration using mature human chondrocyte cells. We compared cell distribution, viability and morphology and we evaluated the mechanical properties of the constructs after 2 weeks of in vitro culture. The materials used as scaffolds were fibrin glue, a collagen sponge and a polyurethane foam (DegraPol(R)). Fibrin glue was found unsuitable as a chondrocyte carrier vehicle after culture times longer than a few days, probably due to significant barriers to nutrients and oxygen diffusion, and the material weakened rapidly. The collagen-based sponge was found to be unsuitable to support chondrocyte survival in vitro, although the presence of newly synthesized collagen was observed in these constructs. The synthetic biodegradable scaffold was more adequate in supporting cell survival and mechanical properties. After 2 weeks of static culture, the storage modulus obtained by dynamic shear testing was in the order of 0.7 kPa in fibrin constructs, 3.7 kPa in collagen constructs and 105 kPa in DegraPol(R) constructs. The better mechanical stability of the synthetic foam supports further investigation in the possible use of synthetic biomaterials as biodegradable scaffolds for in vitro cartilage regeneration. (Journal of Applied Biomaterials & Biomechanics 2004; 2: 55-64).

Elastic stabilization alone or combined with rigid fusion in spinal surgery: a biomechanical study and clinical experience based on 82 cases.

The authors report their experience with the treatment of lumbar instability by a kind of spine stabilization. The elastic stabilization, which follows a new philosophy, is obtained by an interspinous device, and should be used alone in degenerative disc disease, recurrent disc herniation and in very low grade instability, or in association with rigid fusion for the prevention of pathology of the border area. In collaboration with bioengineers, we carried out an experimental study on a lumbar spine model in order to calculate stresses and deformations of lumbar disc during simulation of motion, in physiological conditions and when elastic stabilization is combined with rigid fusion. Results suggest that elastic stabilization reduces stresses on the adjacent disc up to 28 degrees of flexion. Based on this preliminary result, we began to use elastic stabilization alone or combined with fusion in 1994. To date, we have performed 82 surgical procedures, 57 using stabilization alone and 25 combined with fusion, in patients affected by degenerative disc disease, disc herniation, recurrence of disc herniation or other pathologies. Clinical results are satisfactory, especially in the group of patients affected by recurrent disc herniation, in whom the elastic device was used alone.

Mechanobiology of engineered cartilage cultured under a quantified fluid-dynamic environment.

Natural cartilage remodels both in vivo and in vitro in response to mechanical forces and hence mechanical stimulation is believed to have a potential as a tool to modulate extra-cellular matrix synthesis in tissue-engineered cartilage. Fluid-induced shear is known to enhance chondrogenesis on animal cells. A well-defined hydrodynamic environment is required to study the biochemical response to shear of three-dimensional engineered cell systems. We have developed a perfused-column bioreactor in which the culture medium flows through chondrocyte-seeded porous scaffolds, together with a computational fluid-dynamic model of the flow through the constructs' microstructure. A preliminary experiment of human chondrocyte growth under static versus dynamic conditions is described. The median shear stress imposed on the cells in the bioreactor culture, as predicted by the CFD model, is 3 x 10(-3) Pa (0.03 dyn/cm(2)) at a flow rate of 0.5 ml/min corresponding to an inlet fluid velocity of 44.2 mum/s. Providing a fluid-dynamic environment to the cells yielded significant differences in cell morphology and in construct structure.

Improved mathematical model of the wear of the cup articular surface in hip joint prostheses and comparison with retrieved components.

This paper presents an analytical model of the cobalt-based alloy-ultra-high molecular weight polyethylene (UHMWPE) wear coupling. Based on a previous model in which the cup wear volume over a gait cycle (WG) was calculated under the simplifying assumption of an ideal rigid coupling, the current version proposes a more realistic wear simulation. All three components of the hip loading force were considered for the contact pressure calculation and all three components of the hip motion were taken into account for the sliding distance calculation. The contact pressure distribution was calculated on the basis of the Hertzian theory for the elastic contact of two bodies with non-conforming geometrical shapes. The wear factor was taken from hip simulator wear tests. The calculated WG is 67 x 10(-6) mm3 for a standard reference patient. The parametric model simulations show that WG increases linearly with the patient weight, femoral head diameter and surface roughness. It increases non-linearly to a maximum and decreases to an asymptotic value with increasing cup/head clearance and with cup isotropic elastic modulus. The cup orientation in the pelvis affects only slightly the total amount of WG whereas it is the dominant factor affecting the shape of the wear distribution. The iso-wear maps show paracentral patterns at low cup inclination angles and marginal patterns at higher inclination angles. The maximum wear depth is supero-posterior when the cup is in neutral alignment and supero-anterior at increasing anteversion angles. Complex patterns with a combination of paracentral and marginal wear were obtained at specific clearance values and cup orientations. The results of the simulations are discussed in relation to the wear distribution measured on the articular surface of 12 UHMWPE components retrieved from failed hip joint prostheses, after a period of in situ functioning.

Quantitative evaluation of the prosthetic head damage induced by microscopic third-body particles in total hip replacement.

The increase of the femoral head roughness in artificial hip joints is strongly influenced by the presence of abrasive particulate entrapped between the articulating surfaces. The aim of the present study is to evaluate the dependence of such damage on the geometry of the particles entrapped in the joint, with reference to the UHMWPE/chrome-cobalt coupling. Five chrome-cobalt femoral heads and their coupled UHMWPE acetabular cups, retrieved at revision surgery after a short period of in situ functioning, have been investigated for the occurrence of third-body damage. This was found on all the prosthetic heads, where the peak-to-valley height of the scratches, as derived from profilometry evaluations, ranged from 0.3-1.3 microm. The observed damage has been divided into four classes, related to the particle motion while being embedded into the polymer. Two kinds of particle morphology have been studied, spherical and prismatic, with size ranging from 5-50 microm. In order to provide an estimation of the damage induced by such particles, a finite element model of the third-body interaction was set up. The peak-to-valley height of the impression due to the particle indentation on the chrome-cobalt surface is assumed as an index of the induced damage. The calculated values range from 0.1-0.5 microm for spherical particles of size ranging from 10-40 microm. In the case of prismatic particles, the peak-to-valley height can reach 1.3 microm and depends both on the size and width of the particle's free corner, indenting the chrome-cobalt. As an example, a sharp-edged particle of size 30 microm can induce on the chrome-cobalt an impression with peak-to-valley height of 0.75 microm, when embedded into the polyethylene with a free edge of 5 microm facing the metallic surface. Negligible damage is induced, if a free edge of 7.5 microm is indenting the counterface. Our findings offer new support to the hypothesis that microscopic third-body particles are capable of causing increased roughening of the femoral head and provide a quantitative evaluation of the phenomenon.

The in-vivo wear performance of prosthetic femoral heads with titanium nitride coating.

This paper reports the study performed on four titanium nitride (TiN) coated prosthetic femoral heads collected at revision surgery together with patient data. Surface topology has been examined using Scanning Electron Microscopy (SEM) and elemental analysis of both coating and substrate have been evaluated using energy-dispersive X-ray spectrometry. Quantitative assessment of the surface topography is achieved using contacting profilometry. The average Ra roughness value is calculated at five different locations for each femoral head. The UHMWPE counterface worn volume has been measured directly on the acetabular components. TiN fretting and coating breakthrough occurred in two of the four components examined. In the damaged coating areas the surface profile is macroscopically saw-toothed with average tooth height 1.5 microm. The average Ra value is 0.02 microm on the undamaged surfaces and 0.37 microm on the damaged ones. Failure of the coating adhesion resulted in the release of TiN fragments and of metallic particulate from the substrate fretting corrosion and in the increase of the head surface roughness affecting counterface debris production. Our results suggest that TiN-coated titanium alloy femoral heads are inadequate in the task of resisting third body wear mechanisms in vivo.