Experimental study of eigenstrains in temporomandibular joint discs using digital image analysis.

The temporomandibular joint is one of the most frequently used joints of the human body. Its malfunction can severely influence patient's well-being. Since the temporomandibular joint disc plays a major role in its functioning, especially in load distribution within the joint, it appears to be a crucial element to understand. This paper aims to improve understanding of the tissues within close in vivo conditions (i.e. hydrated at 37 ° C) by (i) comforting literature by revealing the presence of residual stresses within the temporomandibular joint disc, (ii) quantifying eigenstrains through a relaxation process and finally (iii) evaluating the internal mechanical state in intact temporomandibular joint discs central part, considering the tissue as a thin layer. Both global specimen size measurements and local digital image correlation were used to quantify 6 samples' deformation through a detailed analysis of approximately 30 images, recorded for approximately one hour, per disc. Thanks to a backward time approach combined to an analytical model, eigenstrains were assessed on discs. For the first time, the presence of complex initial strain fields within cylindrical specimens of porcine temporomandibular joint discs was quantified, confirming indications from literature. Digital image analysis revealed the partial internal stress release through specimen self-deformation. Close to zero in central part, it reached approximately 13% radial strain in the outer ring within a characteristic relaxation time close to 530s. The principal strains' distribution agrees with the alignment of the collagen fibers in the central part of the discs revealed in many works. It led to deduce that, in the central area of the discs, the matrix undergoes a radial compression within physiological conditions to compensate the daily loading stresses. Therefore, this work improves understanding of the tissues in vivo conditions highlighting extraction cut effect on temporomandibular joint disc's tissues mechanical state.

A new method to estimate 3D cell parameters from 2D microscopy images.

Optical microscopy has been a basic and standard technique in cell biology research for decades. Microscopy techniques function well for thin, optically transparent cultures and allow for the imaging of thicker biological specimens. There is no better method of in vitro cell observation and analysis, hence microscopic techniques are extensively used and constitute an optimal tool for cell culture studies. This paper proposes an original methodology of optical microscopy data processing based on the phase contrast technique during cell culture monitoring. By exploiting images recorded during cell proliferation, a surface reconstruction was performed based on assumption, it can be considered that the local brightness of the image depends on the cells' thickness and thus the obtained results can be interpreted in the form of a surface that represents a three-dimensional structure, which allowed for a quantitative description of the cell evolution. The 3D data obtained enabled the investigation of parameters describing the morphology of the cells and the topology of their proliferation. These parameters included cell sizes in plane but also in the direction perpendicular to it, cell volume changes, their spatial distribution, as well as anisotropy and directivity. The method presented provides data carrying information similar to that obtained using a holographic microscope, e.g. A HoloMonitor (Phase Holographic Imaging PHI Inc.), or from confocal scanning microscopy with the "z-stack" mode. The techniques of bright field or phase contrast cell observation are, however, much cheaper, and widely available when compared to holographic microscopy, for instance. Besides, these also enable monitoring of cell activity over time, i.e. the study and quantitative description of dynamic changes in the cells. The proposed approach uses generally available free tools such as ImageJ software with BoneJ and Particle Analyzer plugins. The methodology is suitable for even a basic microscope, it can be easily implemented as a script, and thus data processing can be significantly shortened, the methodology can be automated, and also applied for data processing in real time.

Molecular machinery providing copper bioavailability for spermatozoa along the epididymial tubule in mouse.

Progressive functional maturation of spermatozoa is completed during the transit of these cells through the epididymis, a tubule structure connecting a testicle to a vas deferens. Epididymal epithelial cells by means of their secretory and absorptive functions determine a highly specialized luminal microenvironment containing multiple organic and inorganic components. The latter include copper ions, which due to their redox properties are indispensable for critical homeostatic processes occurring in spermatozoa floating in different part of epididymis but can be potentially toxic. Main purpose of our study was to determine epididymal region-dependent expression and localization of copper transporters ensuring a tight control of copper concentration in epididymal fluid. We also aimed at identifying proteins responsible for copper uptake by spermatozoa and verifying whether this process is coordinated with copper supply to superoxide dismutase 1 (SOD1), a copper-dependent antioxidant enzyme. Our study identifies two ATPases-ATP7A, ATP7B and Slc31a1, major copper importers/exporters depending on their differential expression on epididymal polarized epithelial cells of the caput, corpus, and cauda. Next, ceruloplasmin seems to be a chief protein transporting copper in the epididymal fluid and providing this biometal to spermatozoa. The entry of copper to germ cells is mediated by Slc31a1 and is correlated with both expressions of copper chaperone for superoxide dismutase (CCS), copper chaperone directly providing copper ions to SOD1 and with the expression and activity of the latter. Our results outline a network of cooperating copper binding proteins expressed in epididymal epithelium and in spermatozoa that orchestrate bioavailability of this microelement for gametes and protect them against copper toxicity.

Fatigue behavior of thin-walled grade 2 titanium samples processed by selective laser melting. Application to life prediction of porous titanium implants.

Because of its biocompatibility and high mechanical properties, the commercially pure grade 2 titanium (CPG2Ti) is largely used for fabrication of patient specific implants or hard tissue substitutes with complex shape. To avoid the stress-shielding and help their colonization by bone, prostheses with a controlled porosity are designed. The selective laser melting (SLM) is well adapted to manufacture such geometrically complicated structures constituted by struts with rough surfaces and relatively small diameters. Few studies were dedicated to characterize the fatigue properties of SLM processed samples and bulk parts. They followed conventional or standard protocols. The fatigue behavior of standard samples is very different from the one of porous raw structures. In this study, the SLM made "as built" (AB) and "heat treated" (HT) tubular samples were tested in fatigue. Wöhler curves were determined in both cases. The obtained endurance limits were equal to σD(AB)=74.5MPa and σD(HT)=65.7MPa, respectively. The heat treatment worsened the endurance limit by relaxation of negative residual stresses measured on the external surface of the samples. Modified Goodman diagram was established for raw specimens. Porous samples, based on the pattern developed by Barbas et al. (2012), were manufactured by SLM. Fatigue tests and finite element simulations performed on these samples enabled the determination of a simple rule of fatigue assessment. The method based on the stress gradient appeared as the best approach to take into account the notch influence on the fatigue life of CPG2Ti structures with a controlled porosity. The direction dependent apparent fatigue strength was found. A criterion based on the effective, or global, nominal stress was proposed taking into account the anisotropy of the porous structures. Thanks to this criterion, the usual calculation methods can be used to design bone substitutes, without a precise modelling of their internal fine porosity.

Genetic algorithms as a useful tool for trabecular and cortical bone segmentation.

The aim of this study was to find a semi-automatic method of bone segmentation on the basis of computed tomography (CT) scan series in order to recreate corresponding 3D objects. So, it was crucial for the segmentation to be smooth between adjacent scans. The concept of graphics pipeline computing was used, i.e. simple graphics filters such as threshold or gradient were processed in a manner that the output of one filter became the input of the second one resulting in so called pipeline. The input of the entire stream was the CT scan and the output corresponded to the binary mask showing where a given tissue is located in the input image. In this approach the main task consists in finding the suitable sequence, types and parameters of graphics filters building the pipeline. Because of the high number of desired parameters (in our case 96), it was decided to use a slightly modified genetic algorithm. To determine fitness value, the mask obtained from the parameters found through genetic algorithms (GA) was compared with those manually prepared. The numerical value corresponding to such a comparison has been defined by Dice's coefficient. Preparation of reference masks for a few scans among the several hundreds of them was the only action done manually by a human expert. Using this method, very good results both for trabecular and cortical bones were obtained. It has to be emphasized that as no real border exists between these two bone types, the manually prepared reference masks were quite conventional and therefore charged with errors. As GA is a non-deterministic method, the present work also contains a statistical analysis of the relations existing between various GA parameters and fitness function. Finally the best sets of the GA parameters are proposed.

Development and mechanical characterization of porous titanium bone substitutes.

Commercially Pure Porous Titanium (CPPTi) can be used for surgical implants to avoid the stress shielding effect due to the mismatch between the mechanical properties of titanium and bone. Most researchers in this area deal with randomly distributed pores or simple architectures in titanium alloys. The control of porosity, pore size and distribution is necessary to obtain implants with mechanical properties close to those of bone and to ensure their osseointegration. The aim of the present work was therefore to develop and characterize such a specific porous structure. First of all, the properties of titanium made by Selective Laser Melting (SLM) were characterized through experimental testing on bulk specimens. An elementary pattern of the porous structure was then designed to mimic the orthotropic properties of the human bone following several mechanical and geometrical criteria. Finite Element Analysis (FEA) was used to optimize the pattern. A porosity of 53% and pore sizes in the range of 860 to 1500 µm were finally adopted. Tensile tests on porous samples were then carried out to validate the properties obtained numerically and identify the failure modes of the samples. Finally, FE elastoplastic analyses were performed on the porous samples in order to propose a failure criterion for the design of porous substitutes.

Biomechanical study of mandible bone supporting a four-implant retained bridge: finite element analysis of the influence of bone anisotropy and foodstuff position.

PURPOSE: The aim of this work was to study the biomechanical behavior of a "All-on-four" implant-supported prosthesis through a finite element analysis using either isotropic or anisotropic properties of bone. The influence of foodstuff position during mastication was also analysed. MATERIALS AND METHODS: A three-dimensional finite element model of a mandible with a prosthesis supported by four implants was developed. The geometry of the edentulous mandible and prosthesis was generated from computed tomography. Four MKIII implants (two vertical and two tilted) were modeled. The bone elastic properties used in the anisotropic simulations were orthotropic. The comparison of isotropic and anisotropic models was carried out in the loading condition of mastication with a foodstuff positioned on molar. Three distinct configurations, corresponding to three foodstuff positions, were then studied. MSC/Marc code was used to perform all computations. RESULTS: Significant differences in stress, strain, and strain energy densities were found in the comparison of isotropic and orthotropic models. Molar position was revealed to be the most critical one, from a stress and strain level point of view, for implants and framework and consequently for peri-implant bone. It was also observed that implant tilting leads to high stress concentrations in bone for the "All-on-four" concept. CONCLUSION: This study showed that the anisotropic behaviour of bone cannot be neglected in the numerical simulations. The actual design of the prosthesis is not optimal concerning the capacity of all metallic parts to support loads. Finally, it was demonstrated that the tilting of implants induced a high stress level at bone-implant interface.

A complete finite element model of a mandibular implant-retained overdenture with two implants: comparison between rigid and resilient attachment configurations.

PURPOSE: The aim of this study was to evaluate the influence of the retention mechanism on the behavior of a mandibular implant-retained overdenture (IRO) during the simulation of mastication. Therefore, a complete three-dimensional finite element model of a mandible with its IRO was developed. MATERIALS AND METHODS: The geometry of the edentulous mandible and overdenture was generated from computed tomography. Two MKIII implants (Nobel Biocare) with ball abutments and Dalbo Plus (Cendres et Métaux) attachments were placed in the canine areas. Three foodstuff positions were analyzed for two retention mechanisms, "resilient" or "rigid". Special attention was given to the modeling of the mandibular environment and of the existing contact between the different components. A probable muscular action was determined following the minimal work principle. RESULTS: The food-crushing force was provided by masseters with a two-third/one-third ratio between working and non-working sides. The "resilient" configuration provided a wider contact area between the mucosa of the denture bearing area and the prosthesis. An increase of the mastication force transiting through the mucosa was also noted and lower stresses were observed in the bone surrounding implants. CONCLUSION: Resilient attachments allowed for an increase of the mastication load transiting through denture bearing surface. Furthermore, this study proposed an accurate model of the mandibular IRO, including its environment and faithful behavior reproduction.

Biomechanical study of a prosthetic solution based on an angled abutment: case of upper lateral incisor.

OBJECTIVES: To study the complex behaviour of an upper lateral incisor restoration using an angled abutment, a mechanical analysis of the abutment bearing capacity was firstly carried out. The evolution of bone properties around implant was then simulated as a function of time to estimate the maximal load that could be supported by the prosthetic solution without bone damage. MATERIALS AND METHODS: According to the Food and Drug Administration procedure, experimental tests were firstly carried out on five samples. A 25 degrees -angled abutment screwed to an implant embedded into a massive steel bloc was submitted to a static loading. Two finite element models were also built: the first one (I) to interpret and complete the results obtained in the experimental part and the second one (II) to simulate bone remodelling around implant considering a strain energy stimulus. RESULTS: According to experiments, the abutment straightening was observed for an average force of 869N. Numerical model (I) confirmed this result and indicated that the initial irreversible deformation (yielding) of abutment was obtained for a 283N compressive force. It could thus be deduced that this abutment can safely be used for an incisor restoration. Model (II) showed that, after 26 months, some of the cancellous bone initially present in an approximately one millimetre thick shell surrounding the implant had reached the density of cortical bone. A safe load notion corresponding to the force leading to the maximal admissible strain value for trabecular bone was introduced. It evolved from 44N after surgery to approximately 160N after 26 months.

Intracellular iron status as a hallmark of mammalian cell susceptibility to oxidative stress: a study of L5178Y mouse lymphoma cell lines differentially sensitive to H(2)O(2).

The redox properties of iron make this metal a key participant in oxygen-mediated toxicity. Accordingly, L5178Y (LY) mouse lymphoma cell lines, which display a unique inverse cross-sensitivity to ionizing radiation (IR) and hydrogen peroxide (H(2)O(2)), are a suitable model for the study of possible differences in the constitutive control of intracellular iron availability. We report here that the level of iron in the cytosolic labile iron pool (LIP), ie, potentially active in the Fenton reaction, is more than 3-fold higher in IR-resistant, H(2)O(2)-sensitive (LY-R) cells than in IR-sensitive, H(2)O(2)-resistant (LY-S) cells. This difference is associated with markedly greater content of ferritin H-subunits (H-Ft) in LY-S than in LY-R cells. Our results show that different expression of H-Ft in LY cells is a consequence of an up-regulation of H-Ft mRNA in the LY-S mutant cell line. In contrast, posttranscriptional control of iron metabolism mediated by iron-responsive element-iron regulatory proteins (IRPs) interaction is similar in the 2 cell lines, although IRP1 protein levels in iron-rich LY-R cells are twice those in iron-deficient LY-S cells. In showing that LY cell lines exhibit 2 different patterns of intracellular iron regulation, our results highlight both the role of high LIP in the establishment of pro-oxidant status in mammalian cells and the antioxidant role of ferritin. (Blood. 2000;95:2960-2966)

Antagonistic action of IFN-beta and IFN-gamma on high affinity Fc gamma receptor expression in healthy controls and multiple sclerosis patients.

Monocyte-macrophage activation by IFN-gamma is characterized by a pronounced increase of high affinity Fc receptors for IgG (Fc gamma RI), capable of triggering respiratory burst, phagocytosis, Ab-dependent cytotoxicity, and release of proinflammatory cytokines. In view of the antagonism of IFN-beta on IFN-gamma action, of interest in the chronic inflammatory disorder multiple sclerosis, we examined the possible effect of IFN-beta on IFN-gamma induction of Fc gamma RI gene expression. We found that IFN-beta significantly down-regulated IFN-gamma-induced Fc gamma RI surface expression in peripheral blood monocytes from healthy donors, in a dose- and time-dependent manner. This down-regulation of Fc gamma RI surface levels did not correspond to a decrease in Fc gamma RI mRNA, suggesting a posttranscriptional effect of IFN-beta. Down-regulation of Fc gamma RI surface expression correlated with diminished cellular signaling through Fc gamma RI, since the IFN-gamma-induced increase in Fc gamma receptor-triggered respiratory burst was nearly completely abrogated by simultaneous addition of IFN-beta. Finally, the same antagonism between both IFNs on Fc gamma RI surface expression was observed in peripheral blood monocytes derived from multiple sclerosis patients; inhibition by IFN-beta was even increased (82+/-11%), as compared with healthy controls (67+/-4%). These results may partially help explain the beneficial effect of IFN-beta in multiple sclerosis.

Antibacterial system generated by lactoferrin in mice in vivo is primarily a killing system.

A single dose of bovine lactoferrin (BLF) was given intravenously (i.v.) to CFW mice 24 hours (h) prior to the i.v. injection of the E. coli lethal dose (LD100). BLF strongly accelerated the clearance rate of E. coli from the blood as well as its killing rate in the liver, lungs, spleen and kidney. The highest clearing and killing rate was found 5 h after E. coli LD100 injection. The most intensive killing in the organs examined was found in the lungs and kidney. Analysis of organs of i.v. BLF-stimulated mice which survived up to day 30 after the infection by E. coli showed that not all animals were definitely pathogen-free. It was concluded that the defense system generated by BLF in mice in vivo is primarily a bacteria-killing one. The participation and cooperation of reticulo-endothelial (RE)-macrophages and granulocytes in the phagocytosis and killing of E. coli may thus be related to the protective activity of LF.

Horse spleen ferritin inhibits superoxide production by equine blood monocytes in vitro.

The effect of horse spleen ferritin (HFR) on the production of superoxide anion (O2.-) by equine blood monocytes was investigated. Preincubation of monocytes with HFR resulted in pronounced inhibition of O2.- production in response to phorbol 12-myristate 13-acetate (PMA), N-formyl-methionyl-leucyl-phenylalanine (FMLP), and opsonized zymosan (OZ). The inhibitory effect of HFR upon stimulation of monocytes with PMA was both dose and time dependent. Maximum inhibition (90%) was observed after preincubation of monocytes with HFR (2 mg/ml) for 18 h before stimulation with PMA. ApoHFR at the same concentration showed only about one-third of the inhibitory effect of iron-saturated HFR. Various iron complexes, such as iron dextran, hemin, or ferric ammonium sulfate, had no significant effect on O2.- production by monocytes. Neither catalase (Cat) nor desferrioxamine (DFO) changed the inhibitory effect of HFR. These findings suggest that HFR may play an important role in inhibition of superoxide generation by equine monocytes. Although the mechanism of this inhibition remains unknown, the results obtained suggest that it is not due to ferritin-dependent oxidative inactivation of the NADPH-oxidase system in stimulated monocytes.

Protective effect of tissue ferritins in experimental Escherichia coli infection of mice in vivo.

The effect of ferritins from horse (FH) and bovine (FB) spleen and murine liver (FM) on the survival rate of CFW mice lethally infected with Escherichia coli (strain 8440-78 K 80/B) was evaluated. Ferritins given intravenously 24 h before intravenous inoculation of bacteria, protected mice most effectively from death due to infection. The effect was dose dependent. At 500 micrograms of ferritin per mouse, the maximum survival rates were 86% (FH), 81% (FM) and 79% (FB), while only 5% of the control mice survived up to the 30th day. The survival rates of animals injected with bovine serum albumin (BSA) and heat-inactivated FB were 8 and 25%, respectively. Intraperitoneal injection of FB was as effective as intravenous in enhancing the resistance of mice against bacteria. These data provide evidence for the beneficial role of tissue ferritins in nonspecific antibacterial resistance.

Lactoferrin can protect mice against a lethal dose of Escherichia coli in experimental infection in vivo.

Experiments were undertaken to demonstrate and partially explain the protective effect of bovine lactoferrin (LB) when administered intravenously to mice 24 h before a challenge with a lethal dose of Escherichia coli. About 70% of mice pretreated with LB survived challenge. The survival rates in control mice treated with E. coli alone and pretreated with bovine serum albumin (BSA), were 4 and 8%, respectively. Human lactoferrin (LH) had almost the same protective effect as LB. Sufficient amounts of ferric ions were given to mice, in single and multiple doses, for full serum transferrin saturation 30 min before or after E. coli administration. The multiple dose of ferric ions did not change considerably the survival rate of mice pretreated with LB. In contrast, a single dose of ferric ions gradually decreased the survival rate of the mice after the first week of experiment. From day 14 this decrease was statistically significant in all groups of mice treated with a single dose of ferric ions when compared with mice pretreated only with LB, and the difference ranged from 25 to 35% on day 30. The possible mechanism(s) of protective effect of LB and role of iron ions are discussed.