Complex deformation of cartilage micropellets following mechanical stimulation promotes chondrocyte gene expression.

BACKGROUND: Articular cartilage (AC)'s main function is to resist to a stressful mechanical environment, and chondrocytes are responding to mechanical stress for the development and homeostasis of this tissue. However, current knowledge on processes involved in response to mechanical stimulation is still limited. These mechanisms are commonly investigated in engineered cartilage models where the chondrocytes are included in an exogeneous biomaterial different from their natural extracellular matrix. The aim of the present study is to better understand the impact of mechanical stimulation on mesenchymal stromal cells (MSCs)-derived chondrocytes generated in their own extracellular matrix. METHODS: A fluidic custom-made device was used for the mechanical stimulation of cartilage micropellets obtained from human MSCs by culture in a chondrogenic medium for 21 days. Six micropellets were positioned into the conical wells of the device chamber and stimulated with different signals of positive pressure (amplitude, frequency and duration). A camera was used to record the sinking of each micropellet into their cone, and micropellet deformation was analyzed using a finite element model. Micropellets were harvested at different time points after stimulation for RT-qPCR and histology analysis. RESULTS: Moderate micropellet deformation was observed during stimulation with square pressure signals as mean von Mises strains between 6.39 and 14.35% were estimated for amplitudes of 1.75-14 kPa superimposed on a base pressure of 50% of the amplitude. The compression, tension and shear observed during deformation did not alter micropellet microstructure as shown by histological staining. A rapid and transient increase in the expression of chondrocyte markers (SOX9, AGG and COL2B) was measured after a single 30-min stimulation with a square pressure signal of 3.5 kPa amplitude superimposed on a minimum pressure of 1.75 kPa, at 1 Hz. A small change of 1% of cyclical deformations when using a square pressure signal instead of a constant pressure signal induced a fold change of 2 to 3 of chondrogenic gene expression. Moreover, the expression of fibrocartilage (COL I) or hypertrophic cartilage (COL X, MMP13 and ADAMTS5) was not significantly regulated, except for COL X. CONCLUSIONS: Our data demonstrate that the dynamic deformation of cartilage micropellets by fluidic-based compression modulates the expression of chondrocyte genes responsible for the production of a cartilage-like extracellular matrix. This lays the foundations for further investigating the chondrocyte mechanobiology and the cartilage growth under mechanical stimulation.

Cartilage biomechanics: From the basic facts to the challenges of tissue engineering.

Articular cartilage (AC) is the thin tissue that covers the long bone ends in the joints and that ensures the transmission of forces between adjacent bones while allowing nearly frictionless movements between them. AC repair is a technologic and scientific challenge that has been addressed with numerous approaches. A major deadlock is the capacity to take in account its complex mechanical properties in repair strategies. In this review, we first describe the major mechanical behaviors of AC for the non-specialists. Then, we show how researchers have progressively identified specific mechanical parameters using mathematical models. There are still gaps in our understanding of some of the observations concerning AC biomechanical properties, particularly the differences in extracellular matrix stiffness measured at the microscale and at the millimetric scale. Nevertheless, for bioengineering applications, AC repair strategies must take into account what are commonly considered the main mechanical features of cartilage: its ability to withstand high stresses through three main behaviors (elasticity, poroelasticity and swelling). Finally, we emphasize that future studies need to investigate AC mechanical properties at different scales, particularly the gradient of mechanical properties around cells and across the cartilage depth, and the differences in mechanical properties at different scales. This multi-scale approach could greatly enhance the success of AC restorative approaches.

Impact of extracellular matrix and collagen network properties on the cervical intervertebral disc response to physiological loads: A parametric study.

Current intervertebral disc finite element models are hard to validate since they describe multi-physical phenomena and contain a huge number of material properties. This work aims to simplify numerical validation/identification studies by prioritizing the sensitivity of intervertebral disc behavior to mechanical properties. A 3D fiber-reinforced hyperelastic model of a C6-C7 intervertebral disc is used to carry out the parametric study. 10 parameters describing the extracellular matrix and the collagen network behaviors are included in the parametric study. The influence of varying these parameters on the disc response is estimated during physiological movements of the head, including compression, lateral bending, flexion, and axial rotation. The obtained results highlight the high sensitivity of the disc behavior to the stiffness of the annulus fibrosus extracellular matrix for all the studied loads with a relative increase in the disc apparent stiffness by 67% for compression and by 57% for axial rotation when the annulus stiffness increases from 0.4 to 2 MPa. It is also shown that varying collagen network orientation, stiffness, and stiffening in the studied configuration range have a noticeable effect on rotational motions with a relative apparent stiffness difference reaching 6.8%, 10%, and 22%, respectively, in lateral bending. However, the collagen orientation does not affect disc response to axial load.

Flip-flop method: A new T1-weighted flow-MRI for plants studies.

The climate warming implies an increase of stress of plants (drought and torrential rainfall). The understanding of plant behavior, in this context, takes a major importance and sap flow measurement in plants remains a key issue for plant understanding. Magnetic Resonance Imaging (MRI) which is well known to be a powerful tool to access water quantity can be used to measure moving water. We describe a novel flow-MRI method which takes advantage of inflow slice sensitivity. The method involves the slice selectivity in the context of multi slice spin echo sequence. Two sequences such as a given slice is consecutively inflow and outflow sensitive are performed, offering the possiblility to perform slow flow sensitive imaging in a quite straigthforward way. The method potential is demonstrated by imaging both a slow flow measurement on a test bench (as low as 10 µm.s-1) and the Poiseuille's profile of xylemian sap flow velocity in the xylematic tissues of a tomato plant stem.

Experimental and numerical identification of cortical bone permeability.

Bone is a complex system, and could be modeled as a poroelastic media. The aim of this paper is to identify the macroscopic value of the cortical bone permeability coefficient. A simple experimental method was designed in order to determine the permeability coefficient. Two bone samples taken from different ox femurs were filled with water, to place them under internal pressure. The measurements gave both the fluid flow through the lateral surfaces and the internal pressure. The originality of this work is the coupling between an experimental process and a structural computation performed with a finite element method. The mean cortical bone permeability coefficient identified was about k=1.1x10(-13)m(2). This value tends to confirm other values found in the literature, obtained by different methods and often at macroscopic scale. It confirms also the domination of vascular permeability (Haversian and Volkmann's canals).

Frequency response of a viscoelastic tensegrity model: Structural rearrangement contribution to cell dynamics.

In an attempt to understand the role of structural rearrangement onto the cell response during imposed cyclic stresses, we simulated numerically the frequency-dependent behavior of a viscoelastic tensegrity structure (VTS model) made of 24 elastic cables and 6 rigid bars. The VTS computational model was based on the nonsmooth contact dynamics (NSCD) method in which the constitutive elements of the tensegrity structure are considered as a set of material points that mutually interact. Low amplitude oscillatory loading conditions were applied and the frequency response of the overall structure was studied in terms of frequency dependence of mechanical properties. The latter were normalized by the homogeneous properties of constitutive elements in order to capture the essential feature of spatial rearrangement. The results reveal a specific frequency-dependent contribution of elastic and viscous effects which is responsible for significant changes in the VTS model dynamical properties. The mechanism behind is related to the variable contribution of spatial rearrangement of VTS elements which is decreased from low to high frequency as dominant effects are transferred from mainly elastic to mainly viscous. More precisely, the elasticity modulus increases with frequency while the viscosity modulus decreases, each evolution corresponding to a specific power-law dependency. The satisfactorily agreement found between present numerical results and the literature data issued from in vitro cell experiments suggests that the frequency-dependent mechanism of spatial rearrangement presently described could play a significant and predictable role during oscillatory cell dynamics.

Partitioning of cortical and deep cytoskeleton responses from transient magnetic bead twisting.

We attempted to estimate in living adherent epithelial alveolar cells, the degree of structural and mechanical heterogeneity by considering two individualized cytoskeleton components, i.e., a submembranous "cortical" cytoskeleton and a "deep" cytoskeleton (CSK). F-actin structure characterizing each CSK component was visualized from spatial reconstructions at low and high density, respectively, especially in a 10-microm-cubic neighborhood including the bead. Specific mechanical properties (Young elastic and viscous modulus E and n) were revealed after partitioning the magnetic twisting cytometry response using a double viscoelastic "solid" model with asymmetric plastic relaxation. Results show that the cortical CSK response is a faster (tau1 < or = 0.7 s), softer (E1: 63-109 Pa), moderately viscous (n1: 7- 18 Pas), slightly tensed, and easily damaged structure compared to the deep CSK structure which appears slower (tau2 approximately 1/2 min), stiffer (E2: 95-204 Pa), highly viscous (n2: 760-1967 Pa s), more tensed, and fully elastic, while exhibiting a larger stress hardening behavior. Adding drug depolymerizing actin filaments decreased predominantly the deep CSK stiffness. By contrast, an agent altering cell-matrix interactions affected essentially the cortical CSK stiffness. We concluded that partitioning the CSK within cortical and deep structures is largely consistent with their respective functional activities.

Toward a generalised tensegrity model describing the mechanical behaviour of the cytoskeleton structure.

The control of many cell functions including growth, migration and mechanotransduction, depends crucially on stress-induced mechanical changes in cell shape and cytoskeleton (CSK) structure. Quantitative studies have been carried out on 6-bar tensegrity models to analyse several mechanical parameters involved in the mechanical responses of adherent cells (i.e. strain hardening, internal stress and scale effects). In the present study, we attempt to generalize some characteristic mechanical laws governing spherical tensegrity structures, with a view of evaluating the mechanical behaviour of the hierarchical multi-modular CSK-structure. The numerical results obtained by studying four different tensegrity models are presented in terms of power laws and point to the existence of unique and constant relationships between the overall structural stiffness and the local properties (length, number and internal stress) of the constitutive components.

A cellular tensegrity model to analyse the structural viscoelasticity of the cytoskeleton.

This study describes the viscoelastic properties of a refined cellular-tensegrity model composed of six rigid bars connected to a continuous network of 24 viscoelastic pre-stretched cables (Voigt bodies) in order to analyse the role of the cytoskeleton spatial rearrangement on the viscoelastic response of living adherent cells. This structural contribution was determined from the relationships between the global viscoelastic properties of the tensegrity model, i.e., normalized viscosity modulus (eta(*)), normalized elasticity modulus (E(*)), and the physical properties of the constitutive elements, i.e., their normalized length (L(*)) and normalized initial internal tension (T(*)). We used a numerical method to simulate the deformation of the structure in response to different types of loading, while varying by several orders of magnitude L(*) and T(*). The numerical results obtained reveal that eta(*) remains almost independent of changes in T(*) (eta(*) proportional, variant T(*+0.1)), whereas E(*) increases with approximately the square root of the internal tension T(*) (from E(*) proportional, variant T(*+0.3) to E(*) proportional, variant T(*+0.7)). Moreover, structural viscosity eta(*) and elasticity E(*) are both inversely proportional to the square of the size of the structure (eta(*) proportional, variant L(*-2) and E(*) proportional, variant L(*-2)). These structural properties appear consistent with cytoskeleton (CSK) mechanical properties measured experimentally by various methods which are specific to the CSK micromanipulation in living adherent cells. Present results suggest, for the first time, that the effect of structural rearrangement of CSK elements on global CSK behavior is characterized by a faster cellular mechanical response relatively to the CSK element response, which thus contributes to the solidification process observed in adherent cells. In extending to the viscoelastic properties the analysis of the mechanical response of the cellular 30-element tensegrity model, the present study contributes to the understanding of recent results on the cellular-dynamic response and allows to reunify the scattered data reported for the viscoelastic properties of living adherent cells.

Interrelations between elastic energy and strain in a tensegrity model: contribution to the analysis of the mechanical response in living cells.

Interactions between the physical and physiological properties of cellular sub-units result in changes in the shape and mechanical behaviour of living tissues. To understand the mechanotransmission processes, models are needed to describe the complex interrelations between the elements and the cytoskeletal structure. In this study, we used a 30-element tensegrity structure to analyse the influence of the type of loading on the mechanical response and shape changes of the cell. Our numerical results, expressed in terms of strain energy as a function of the overall deformation of the tensegrity structure, suggest that changes in cell functions during mechanical stimuli for a given potential energy are correlated to the type of loading applied, which determines the resultant changes in cell shape. The analysis of these cellular deformations may explain the large variability in the response of bone cells submitted to different types of mechanical loading.

Tensegrity behaviour of cortical and cytosolic cytoskeletal components in twisted living adherent cells.

The present study is an attempt to relate the multicomponent response of the cytoskeleton (CSK), evaluated in twisted living adherent cells, to the heterogeneity of the cytoskeletal structure--evaluated both experimentally by means of 3D reconstructions, and theoretically considering the predictions given by two tensegrity models composed of (four and six) compressive elements and (respectively 12 and 24) tensile elements. Using magnetic twisting cytometry in which beads are attached to integrin receptors linked to the actin CSK of living adherent epithelial cells, we specifically measured the elastic CSK response at quasi equilibrium state and partitioned this response in terms of cortical and cytosolic contributions with a two-component model (i.e., a series of two Voigt bodies). These two CSK components were found to be prestressed and exhibited a stress-hardening response which both characterize tensegrity behaviour with however significant differences: compared to the cytosolic component, the cortical cytoskeleton appears to be a faster responding component, being a less prestressed and easily deformable structure. The discrepancies in elastic behaviour between the cortical and cytosolic CSK components may be understood on the basis of prestress tensegrity model predictions, given that the length and number of constitutive actin elements are taken into account.