Fiber orientation and crimp level might control the auxetic effect of biological tissues.

We propose an analytical micromechanical model for studying the lamellar-composite-like structure of fibrous soft tissue. The tissue under consideration is made up of several lamellae, and is designed to resemble the annulus fibrosus (AF) tissue or media layer of arterial tissue, for example. The collagen fibers are arranged in parallel in each lamella and the fiber orientation differs from one lamella to its neighbors. The parallel fibers in each lamella of AF tissue, for example, have been observed to have a crimped microstructure. The proposed model incorporates this quality, considering fiber waviness as a sinusoidal shape and taking into account the fiber dispersion in different layers, where both fiber and matrix are considered as solid phases. We find that collagen-fiber waviness and layer orientation have a significant influence on Poisson's ratio. The effective Poisson's ratio predicted by the proposed model demonstrates that the crimped collagen fiber microstructure might weaken the auxetic effect of fibrous soft tissue, which might explain why, as the literature suggests, the auxetic behavior is more difficult to observe than large Poisson's ratios. As opposed to the many studies that use the well-known hyperelastic fiber-based constitutive model, in which out-of-plane expansion is often observed, the present work explains the auxetic response found in modeling and in experimental data from the perspective of collagen fiber microstructure.

Validation of a new fluidic device for mechanical stimulation and characterization of microspheres: A first step towards cartilage characterization.

Articular cartilage is made of chondrocytes surrounded by their extracellular matrix that can both sense and respond to various mechanical stimuli. One of the most widely used in vitro model to study cartilage growth is the model of mesenchymal stromal cells-derived cartilage micropellet. However, mechanical stimulation of micropellets has never been reported probably because of their small size and imperfect round shape. The objective of the study was to develop an original custom-made device allowing both the mechanical stimulation and characterization of cartilage micropellets. The fluidic-based device was designed for the concomitant stimulation or characterization of six microspheres placed into the conical wells of a tank. In the present study, the device was validated using alginate-, collagen- and crosslinked collagen-based microspheres. Different types and ranges of pressure signals (square, sinusoidal and constant) were applied. The mechanical properties of microspheres were equivalent to those determined by a conventional compression test. Accuracy, repeatability and reproducibility of all types of pressure signals were demonstrated even though square signals were less accurate and sinusoidal signals were less reproducible than the others. The interest of this new device lies in the reliability to mechanically stimulate and characterize microspheres with diameters in the range of 900 to 1500 µm. Mechanical stimulation can be performed on six microspheres in parallel allowing the mechanical and molecular characterization of the same group of cartilage micropellets. The device will be useful to evaluate the growth of cartilage micropellets under mechanical stimuli.

Mesenchymal stem cells-derived cartilage micropellets: A relevant in vitro model for biomechanical and mechanobiological studies of cartilage growth.

The prevalence of diseases that affect the articular cartilage is increasing due to population ageing, but the current treatments are only palliative. One innovative approach to repair cartilage defects is tissue engineering and the use of mesenchymal stem/stromal cells (MSCs). Although the combination of MSCs with biocompatible scaffolds has been extensively investigated, no product is commercially available yet. This could be explained by the lack of mechanical stimulation during in vitro culture and the absence of proper and stable cartilage matrix formation, leading to poor integration after implantation. The objective of the present study was to investigate the biomechanical behaviour of MSC differentiation in micropellets, a well-defined 3D in vitro model of cartilage differentiation and growth, in view of tissue engineering applications. MSC micropellet chondrogenic differentiation was induced by exposure to TGFß3. At different time points during differentiation (35 days of culture), their global mechanical properties were assessed using a very sensitive compression device coupled to an identification procedure based on a finite element parametric model. Micropellets displayed both a non-linear strain-induced stiffening behaviour and a dissipative behaviour that increased from day 14 to day 29, with a maximum instantaneous Young's modulus of 179.9 ± 18.8 kPa. Moreover, chondrocyte gene expression levels were strongly correlated with the observed mechanical properties. This study indicates that cartilage micropellets display the biochemical and biomechanical characteristics required for investigating and recapitulating the different stages of cartilage development.

Heterogeneous mechanical hyperelastic behavior in the porcine annulus fibrosus explained by fiber orientation: An experimental and numerical approach.

Our aim is to estimate regional mechanical properties of the annulus fibrosus (AF) using a multi-relaxation tensile test and to examine the relevance of using the transverse dilatations in the identification procedure. We collected twenty traction specimens from both outer (n = 10) and inner (n = 10) sites of the anterior quadrant of the annulus fibrosus of one pig spine. A 1-h multi-relaxation tensile test in the circumferential direction allowed us to measure the force in the direction of traction and the dilatations in all three directions. We performed a specific-sample finite element inverse analysis to identify variations, along the radial position, of material and structural parameters of a hyperelastic compressible and anisotropic constitutive law. Our experimental results reveal that the outer sites are subjected to a significantly greater stress than the inner sites and that both sites exhibit an auxetic behavior. Our numerical results suggest that the inhomogeneous behavior arises from significant variations of the fiber angle taken into account within the hyperelastic constitutive law. In addition, we found that the use of the measured transverse dilatations in the identification procedure had a strong impact on the identified mechanical parameters. This pilot study suggests that, in quasi-static conditions, the annulus fibrosus may be modeled by a hyperelastic compressible and anisotropic law with a fiber angle gradient from inner to outer periphery.

Novel endodontic sealers induce cell cytotoxicity and apoptosis in a dose-dependent behavior and favorable response in mice subcutaneous tissue.

OBJECTIVES: The objective of the present study is to evaluate the in vitro cytotoxicity and in vivo biocompatibility of two novel endodontic sealers: RealSeal XT1 and Sealapex Xpress on the subcutaneous connective tissue of mice. MATERIALS AND METHODS: The cytotoxicity was assessed by cell viability using the MTT assay (one-way ANOVA), trypan blue test (Mann-Whitney) and cell apoptosis by flow cytometer. For the subcutaneous study, polyethylene tubes filled with the sealers were implanted in 70 BALB/c mice: 6 experimental groups (n = 10/group) and 2 control groups with empty tubes (n = 5/group). At the end of experimental periods (7, 21, and 63 days), the tissue was removed and histotechnically processed. Angioblastic proliferation and edema (Fisher's exact test) were evaluated, besides thickness measurement (µm) of the reactionary granulomatous tissue and neutrophil counts (Kruskal-Wallis and Dunn's post test; Mann-Whitney) (α = 0.05). RESULTS: MTT assay, trypan blue, and analysis of apoptotic cells showed a dose-dependent direct effect: the more diluted the sealer, the less cytotoxic. Regarding the angioblastic proliferation and edema, difference between the sealers at 7 and 63 days occurred (p < 0.05). Both endodontic sealers initially promoted perimaterial tissue reaction as a foreign body granuloma and thus stimulated favorable tissue responses. CONCLUSIONS: Both sealers showed a dose-dependent effect and promoted satisfactory subcutaneous tissue response; the sealer Sealapex Xpress was less cytotoxic and more biocompatible than RealSeal XT. CLINICAL RELEVANCE: The step of root canal filling during endodontic treatment is highly important for the preservation of the periapical tissue integrity. Subcutaneous reaction to endodontic sealers enables scientific basis for clinical use.

Simulation of cellular packing in non-proliferative epithelia.

The physical laws governing the morphogenesis of biological tissues remain largely misunderstood. In particular, the role of the mechanical interactions occurring in this process needs to be better understood and studied. Inner follicular cells surrounding the oocytes of Ciona intestinalis form an epithelial monolayer resulting from an accretion process (without mitosis or apoptosis). This epithelium is elementary and useful for morphogenesis studies: the cells exhibit polygon packing with a specific but non-systematically repeatable topology (i.e. the distribution of pentagons, hexagons and heptagons changes). To understand the role of mechanical forces in tissue formation, we propose an innovative "2D spherical" model based on the physics of divided media. This approach simulates the cellular mechanical behavior and epithelium structuration by allowing cells to adopt a large variety of shapes and to self-organize in response to mechanical interactions. The numerical parameters considered in the model are derived from experimental data in order to perform pertinent and realistic simulations. The results obtained are compared to biological observations using the same counting method to characterize epithelium topology. Numerical and experimental data appear close enough to validate the model. It is then used for exploratory studies dealing with "Tissue Development Speed" variation, which is not easily attainable by experimentation. We show that the formation speed of the tissue influences its topology and hence its packing organization.

Mechanical model of cytoskeleton structuration during cell adhesion and spreading.

The biomechanical behavior of an adherent cell is intimately dependent on its cytoskeleton structure. Several models have been proposed to study this structure taking into account its existing internal forces. However, the structural and geometrical complexities of the cytoskeleton's filamentous networks lead to difficulties for determining a biologically realistic architecture. The objective of this paper is to present a mechanical model, combined with a numerical method, devoted to the form-finding of the cytoskeleton structure (shape and internal forces) when a cell adheres on a substrate. The cell is modeled as a granular medium, using rigid spheres (grains) corresponding to intracellular cross-linking proteins and distant mechanical interactions to reproduce the cytoskeleton filament internal forces. At the initial state (i.e., before adhesion), these interactions are tacit. The adhesion phenomenon is then simulated by considering microtubules growing from the centrosome towards transmembrane integrin-like receptors. The simulated cell shape changes in this process and results in a mechanically equilibrated structure with traction and compression forces, in interaction with the substrate reactions. This leads to a compressive microtubule network and a corresponding tensile actin-filament network. The results provide coherent shape and forces information for developing a mechanical model of the cytoskeleton structure, which can be exploitable in future biomechanical studies of adherent cells.

Mechanisms governing the visco-elastic responses of living cells assessed by foam and tensegrity models.

The visco-elastic properties of living cells, measured to date by various authors, vary considerably, depending on the experimental methods and/or on the theoretical models used. In the present study, two mechanisms thought to be involved in cellular visco-elastic responses were analysed, based on the idea that the cytoskeleton plays a fundamental role in cellular mechanical responses. For this purpose, the predictions of an open unit-cell model and a 30-element visco-elastic tensegrity model were tested, taking into consideration similar properties of the constitutive F-actin. The quantitative predictions of the time constant and viscosity modulus obtained by both models were compared with previously published experimental data obtained from living cells. The small viscosity modulus values (10(0)-10(3) Pa x s) predicted by the tensegrity model may reflect the combined contributions of the spatially rearranged constitutive filaments and the internal tension to the overall cytoskeleton response to external loading. In contrast, the high viscosity modulus values (10(3)-10(5) Pa x s) predicted by the unit-cell model may rather reflect the mechanical response of the cytoskeleton to the bending of the constitutive filaments and/or to the deformation of internal components. The present results suggest the existence of a close link between the overall visco-elastic response of micromanipulated cells and the underlying architecture.