Hydrated Tissues Research Articles

Uptake and distribution of nickel and other metals in the hyperaccumulator Berkheya coddii

Summary Berkheya coddii is a fast‐growing, high biomass nickel (Ni) hyperaccumulator plant that has recently attracted attention for its possible use in the phytoextraction of Ni. The mechanisms of Ni accumulation, however, are not well understood in this plant. Plants were grown hydroponically in varying Ni concentrations to assess the uptake and distribution of Ni, and other metals, at the whole plant level. X‐ray microanalyses (EDXA) of frozen hydrated tissues were conducted to determine the distribution of Ni at the cellular level in the leaves. Most Ni was found in the shoots, especially in the leaves. Leaf Ni concentration increased with age, whereas older stem sections had lower Ni concentrations than new growth. EDXA analyses revealed that the cuticle of the upper epidermis had a significantly higher Ni concentration than the rest of the leaf. The Ni concentrations in the other leaf tissues were not significantly different. This pattern of distribution contrasts sharply with some other hyperaccumulator species that commonly show a preferential accumulation of Ni and other metals in the vacuoles of the epidermal cells.

INFLUENCE OF MECHANICAL STRESS ON EXTRACELLULAR MATRIXES SYNTHESIZED BY CHONDROCYTES SEEDED ONTO ALGINATE AND HYALURONATE-BASED 3D BIOSYSTEMS

Articular cartilage is an hydrated tissue that withstands and distributes mechanical stresses. The chondrocytes respond to mechanical signals by regulating their metabolic activity through complex biological and biophysical interactions with the extracellular matrix (ECM). The objective of this work was to compare, under mechanical stress, the ECMs synthesized by rat chondrocytes seeded onto biosystems based on alginate (Alg), hyaluronic acid (HA) and a HA amphiphilic derivative. The mechanical stress simulates the traumatisms resulting from accidental shocks or intensive physical exercise by knocking the biosystems together. The investigation of ECMs neosynthesized by chondrocytes was carried out according to various criteria: proliferation, proteoglycans synthesis activity, expression of type I and type II collagens and the expression of α5/β1 integrin. The results obtained for the stress applied on neosynthesized matrixes of 3, 10, 17 and 24 days evidenced a high proliferation and proteoglycans synthesis activity for cells submitted to a knocking process. For all biosystems, the neosynthesized matrix contained an important level of collagen, which was in part of type II, whatever the biosystems. Finally, the chemical modification of HA by long hydrophobic alkyl chains, affords an amphiphilic derivative with viscoelastic properties perfectly mimicking those of matricial environment of chondrocytes. This study showed that the HA amphiphilic derivative induced biological effects similar to those of parent HA containing no hydrophobic modifications.

Modelling of 3‐D Linear Viscoelastic Swelling of Charged Tissues and Gels

AbstractThe skeleton of hydrated tissues or gels exhibits flow‐independent viscoelastic properties [2] which are strongly coupled with the dissipative phenomena resulting from the interstitial fluid flow and the electrochemical swelling mechanisms [4]. Following this, it is the goal of this contribution to combine a linear viscoelasticity formulation with an electrochemical swelling theory in the framework of a well‐founded multiphasic concept. Proceeding from a macroscopic mixture approach, the governing equations can be expressed in terms of three primary variables, namely the solid displacement uS, the effective pore‐fluid pressure p and the molar salt concentration cm of the interstitial fluid.

Fracture-toughening mechanisms responsible for differences in work to fracture of hydrated and dehydrated dentine

This study investigates the nature of deformation and differences in the mechanisms of fracture and properties of dentine where there has been a loss of moisture, as may occur with removal of the pulp in the endodontic treatment of teeth. Controlled fracture toughness testing was conducted on bovine teeth to determine the influence of hydration on the work of fracture of dentine. Significant differences ( p<0.01) were observed between the fracture toughness of hydrated (554±27.7 J/m 2) and dehydrated (113±17.8 J/m 2) dentine. Observations of the crack tip region during crack extension revealed extensive ligament formation occurred behind the crack tip. These ligaments provide considerable stability to the crack by significantly increasing the work of fracture, thereby acting as a fracture-toughening mechanism. Micro-cracking, reported as a fracture-toughening mechanism in bone, is also clearly seen. A zone of in-elastic deformation may occur as hydrated specimens revealed upon crack extension, a region about the tip that appeared to suck water into the structure and to exude water behind the crack tip. In dehydrated dentine, no in-elastic zone was observed. Micro-cracking is present though the cracks are smaller, straighter and with less opening than hydrated dentine. Only limited ligament formation just behind the crack tip was observed. These differences resulted in a significantly lower work of fracture with unstable brittle fracture characteristics. Based on these results, several fracture-toughening mechanisms were identified in dentine, with micro-cracking not considered the most important. These findings may be relevant for bone, a similar mineralised hydrated tissue.

Wire-shaped ultrasonic catheter with diamond coated head for ultrasonic angioplasty

The invention features a wire-shaped ultrasonic catheter (4) for ultra sonic angioplasty, characterised in that the ultrasonic catheter head is provided with a diamond coating (10). The invention provides for greater efficiency for breaking up hard calcified atherosclerosis plaques, without losing its efficacy in breaking up blood clots and other highly hydrated biological tissues, leading to a surgical operation of shorter duration. Further, the invention produces very small-sized debris, when breaking up hard calcified atherosclerosis plaques, which allows for their easy elimination by aspiration or by the natural ducts with respect to angioplasty systems using diamond-coated burs, the ultrasonic catheter with diamond-coated head, due to its longitudinal vibration operating mode, presents substantially reduced risks of damaging the artery wall, and hence greater security for the patient.

Oxalate oxidases and differentiating surface structure in wheat: germins

Oxalate oxidases (OXOs) have been found to be concentrated in the surface tissues of wheat embryos and grains: germin is concentrated in root and leaf sheaths that surround germinated embryos; pseudogermin (OXO-ψ) is concentrated in the epidermis and bracts that ‘encircle’ mature grains. Most strikingly, the epidermal accumulation of OXO-ψ was found to presage the transition of a delicate ‘skin’, similar to the fragile epidermis of human skin, into the tough shell (the miller's ‘beeswing’) that is typical of mature wheat grains. A narrow range of oxalate concentration (1-2 mM) in the hydrated tissues of major crop cereals (barley, maize, oat, rice, rye and wheat) contrasted with wide variations in their OXO expression, e.g. cold-tolerant and cold-sensitive varieties of maize have similar oxalate contents but the former was found to contain approx. 20-fold more germin than did the latter. Well-known OXOs in sorghum, a minor cereal, and beet, a dicotyledon, were found to have little antigenic relatedness to the germins, but the beet enzyme did share some of the unique stability properties that are peculiar to the germin-like OXOs that are found only in the major crop cereals. Their concentration in surface structures of domesticated wheat suggests a biochemical role for germin-like OXOs: programmed cell death in surface tissues might be a constitutive as well as an adaptive form of differentiation that helps to produce refractory barriers against tissue invasion by predators. Incidental to the principal investigation, and using an OXO assay (oxalate-dependent release of CO2) that did not rely on detecting H2O2, which is often fully degraded in cell extracts, it was found that OXO activity in soluble extracts of wheat was manifested only in standard solution assays if the extract was pretreated in a variety of ways, which included preincubation with pepsin or highly substituted glucuronogalactoarabinoxylans (cell-wall polysaccharides).

Oxalate oxidases and differentiating surface structure in wheat: germins.

Oxalate oxidases (OXOs) have been found to be concentrated in the surface tissues of wheat embryos and grains: germin is concentrated in root and leaf sheaths that surround germinated embryos; pseudogermin (OXO-psi) is concentrated in the epidermis and bracts that 'encircle' mature grains. Most strikingly, the epidermal accumulation of OXO-psi was found to presage the transition of a delicate 'skin', similar to the fragile epidermis of human skin, into the tough shell (the miller's 'beeswing') that is typical of mature wheat grains. A narrow range of oxalate concentration (1--2 mM) in the hydrated tissues of major crop cereals (barley, maize, oat, rice, rye and wheat) contrasted with wide variations in their OXO expression, e.g. cold-tolerant and cold-sensitive varieties of maize have similar oxalate contents but the former was found to contain approx. 20-fold more germin than did the latter. Well-known OXOs in sorghum, a minor cereal, and beet, a dicotyledon, were found to have little antigenic relatedness to the germins, but the beet enzyme did share some of the unique stability properties that are peculiar to the germin-like OXOs that are found only in the major crop cereals. Their concentration in surface structures of domesticated wheat suggests a biochemical role for germin-like OXOs: programmed cell death in surface tissues might be a constitutive as well as an adaptive form of differentiation that helps to produce refractory barriers against tissue invasion by predators. Incidental to the principal investigation, and using an OXO assay (oxalate-dependent release of CO(2)) that did not rely on detecting H(2)O(2), which is often fully degraded in cell extracts, it was found that OXO activity in soluble extracts of wheat was manifested only in standard solution assays if the extract was pretreated in a variety of ways, which included preincubation with pepsin or highly substituted glucuronogalactoarabinoxylans (cell-wall polysaccharides).

The acetabular labrum seal: a poroelastic finite element model

Objective. The aim of the study is to investigate the labrum’s ability to seal a pressurised layer of synovial fluid within the joint, and to study the influence of this sealing mechanism on cartilage deformation, interstitial fluid pressure and collagen solid matrix stresses. Background. Cartilage degeneration has been observed in conjunction with labrum pathology. However, little is known about the function of the labrum. Experimental observations have been reported which are consistent with a sealing function of the labrum. Methods. The model was an axisymmetric geometric approximation of the acetabular and femoral cartilage layers and the surrounding labrum. A poroelastic formulation was used to account for the solid and fluid components of these hydrated tissues. A sensitivity analysis of the labrum material properties was carried out. Results. With a compressive load of 1200 N applied across the joint model, the labrum could seal a layer of pressurised fluid between the femur and acetabulum, thus preventing contact of the articulating surfaces. With this sealing effect, loads were transferred across the joint predominantly by uniform pressurisation of the interstitial fluid of the cartilage layers. In the absence of this sealing, strains within the solid matrix of the cartilage layers were higher (e.g. 20% vs. 3%). Conclusions. The labrum can seal against fluid expression from the joint space. This sealing function protects the cartilage layers of the hip. Relevance Current treatments for labrum damage and early arthrosis may compromise the sealing function of the labrum. With continued study of the function and importance of the labrum, new surgical repair strategies can be developed to maintain the overall function of the hip joint.

A new formulation and computation of the triphasic model for mechano-electrochemical mixtures

From the generalized first law of thermodynamics for an irreversible thermodynamical system, a new set of governing equations for the mixture theory is derived based on the triphasic model for mechano-electrochemical mixtures. It is shown that, in the case of electroneutral solution, a new biphasic mixture theory including the electrochemical effects can be derived from the new governing equations. The chemical-expansion stress representing both the influences of deformation on the fixed charge density and the electric potential of fixed charge field is given. For comparison and verification purposes, the numerical solution for a confined compression problem of a charged hydrated soft tissue is computed using the multiquadric method.

Porohyperelastic–transport–swelling theory, material properties and finite element models for large arteries

A porohyperelastic–transport–swelling (PHETS) model is presented in which a soft hydrated tissue material is viewed as a continuum composed of an incompressible porous solid ( fibrous matrix) that is saturated by an incompressible fluid (water) in which a mobile species (solute) is dissolved. This PHETS theoretical model is implemented using a finite element model (FEM) including inherent nonlinearity, coupled transport processes, and complicated geometry and boundary conditions associated with soft tissue structures. The PHETS material properties are clearly identified with a physical basis describing and quantifying elasticity, permeability, diffusion, convection, and osmotic properties. The equivalence between the PHETS and the triphasic (TRI) model (Lai et al., 1991) is established using the phenomenological equations, and mathematical expressions are given to relate the PHETS and TRI material properties. A principle of virtual velocities (PVV ) links Eulerian and Lagrangian PHETS formulations and provides correspondence rules between the Eulerian and the Lagrangian field variables and material properties. The PVV is also the basis for a mixed Lagrangian PHETS FEM (Kaufmann, 1996) , which was developed for the analysis of soft hydrated tissues. Selected PHETS FEM results are presented in order to demonstrate the capability of the PHETS model to simulate coupled deformation, stress, mobile water flux, and albumin flux in the arterial wall undergoing finite straining associated with pressurization, axial stretch, and changes in albumin concentration in bath solutions surrounding a segment of rabbit thoracic aorta. Values for isotropic material parameters and specific details of the experiments and data-reduction methods were obtained from Simon et al. (1997 ; 1998) .

Finite element formulations for hyperelastic transversely isotropic biphasic soft tissues

This paper presents a finite element model for the three-dimensional (3-D) nonlinear analysis of soft hydrated tissues such as articular cartilage in diarthrodial joints under physiologically relevant loading conditions. A biphasic continuum description is used to represent the soft tissue as a two-phase mixture of incompressible inviscid fluid and a hyperelastic, transversely isotropic solid. Alternate mixed-penalty and velocity-pressure finite element formulations are used to solve the nonlinear biphasic governing equations, including the effects of a strain-dependent permeability and a hyperelastic solid phase under finite deformation. The resulting first-order nonlinear system of equations are discretized in time using an implicit finite difference scheme and solved using the Newton-Raphson method. A significant contribution of this work is the implementation and testing of a biphasic description with a transversely isotropic hyperelastic solid phase. This description considers a Helmholtz free energy function of five invariants of the Cauchy-Green deformation tensor and the preferred direction of the material, allowing for asymmetric behavior in tension and compression. An exponential form is suggested and a set of material parameters is identified to represent the response of soft tissues in ranges of deformation and stress observed experimentally. After demonstrating the behavior of this constitutive model in simple tension and compression, a sample problem of unconfined compression is used to further validate the finite element implementation.

A contact finite element formulation for biological soft hydrated tissues

As part of the highly complex numerical simulation of human joints, a finite element formulation for contact between layers of articular cartilage has been developed. A biphasic description of the cartilage is used, where the material is considered to be a porous, permeable solid matrix with fluid flowing through it. Governing equations and contact boundary conditions are summarized for this material before developing the finite element formulation from a Galerkin weighted residual statement. Lagrange multipliers are introduced to enforce the two kinetic continuity equations across the contact surface, and an iterative scheme is employed to determine the current contact area. Numerical examples are presented which demonstrate both that the appropriate continuity across the contact surface is enforced and that the contact surface definition is accurate.

Multiquadric method for the numerical solution of a biphasic mixture model

A computational algorithm based on the multiquadric method has been devised to solve the biphasic mixture model. The model includes a set of constitutive equations for the fluid flows through the solid phase; a set of momentum equations for stress-strain equilibrium and a continuity equation for the solid phase and the fluid phase. The numerical method does not require the generation of mesh as in the finite element method and hence gives high flexibility in applying the method to irregular geometry. Numerical examples are made to compute the solution of the confined compression problem which approximates the nonlinear response of soft hydrated tissues under external loading. The numerical results are compared with Spilker's penalty finite element method.

Importance of the Fixative for Reliable Ultrastructural Preservation of Poikilohydric Plant Tissues. Observations on Dry, Partially, and Fully Hydrated Tissues ofSelaginella lepidophylla

Abstract Leaves of desiccated ‘resurrection plants’, Selaginella lepidophylla , were hydrated either through the roots of intact plants or as isolated organs. Air-dry tissue and samples at 1, 4, 8 and 24 h (both detached and intact) of hydration were prepared for electron microscopy using aldehyde fixatives of different osmotic strengths. Both dry and hydrated tissues were also prepared using freeze substitution. Significant differences in the ultrastructural preservation of these different samples were noted. There was a direct correlation between the osmolality of both the fixative and the tissue with the quality of ultrastructural preservation. When the osmolality of the fixative was slightly (or even considerably) higher than that of the tissue, optimal preservation was achieved. Freeze substitution, however, gave the most faithful preservation of all subcellular compartments, despite the frequent presence of small ice crystals. Additionally, hydration of detached leaves for more than 4 h resulted in swelling damage of the organelles and cytoplasm, regardless of the fixation protocol. Broadly interpreted, the results of this study indicate that an optimal preservation of plant cell and organelle ultrastructure can be achieved by the use of high osmolality fixatives or, preferably, freeze substitution. These results are also important in determining the method of hydration of poikilohydric samples for physiological studies and for interpretation of functional changes as related to the structural condition of the organelles.

Mixed and Penalty Finite Element Models for the Nonlinear Behavior of Biphasic Soft Tissues in Finite Deformation: Part II — Nonlinear Examples

This two-part paper addresses finite element-based computational models for the three-dimensional (3-D) nonlinear analysis of soft hydrated tissues, such as articular cartilage in diarthrodial joints, under physiologically relevant loading conditions. A biphasic continuum description is used to represent the soft tissue as a two-phase mixture of incompressible inviscid fluid and a hyperelastic, transversely isotropic solid. Alternate mixed-penalty and velocity-pressure finite element formulations are used to solve the nonlinear biphasic governing equations, including the effects of strain-dependent permeability and a hyperelastic solid phase under finite deformation. The resulting first-order, nonlinear system of equations is discretized in time using an implicit finite difference scheme, and solved using the Newton-Raphson method. Details of the formulations were presented in Part 1 [I], In Part II, the two formulations are used to develop two-dimensional (2-D) quadrilateral and triangular elements and three-dimensional (3-D) hexahedral and tetrahedral elements. Numerical examples, including those representative of soft tissue material testing and simple human joints, are used to validate the formulations and to illustrate their applications. A focus of this work is the comparison of the alternate formulations for nonlinear problems. While it is demonstrated that both formulations produce a range of converging elements, the velocity-pressure formulation is found to be more efficient computationally.

Mixed and Penalty Finite Element Models for the Nonlinear Behavior of Biphasic Soft Tissues in Finite Deformation: Part I — Alternate Formulations

This paper addresses finite element-based computational models for the three-dimensional, (3-D) nonlinear analysis of soft hydrated tissues, such as the articular cartilage in diarthrodial joints, under physiologically relevant loading conditions. A biphasic continuum description is used to represent the soft tissue as a two-phase mixture of incompressible, inviscid fluid and a hyperelastic solid. Alternate mixed-penalty and velocity-pressure finite element formulations are used to solve the nonlinear biphasic governing equations, including the effects of a strain-dependent permeability and a hyperelastic solid phase under finite deformation. The resulting first-order nonlinear system of equations is discretized in time using an implicit finite difference scheme, and solved using the Newton-Raphson method. Using a discrete divergence operator, an equivalence is shown between the mixed-penalty method and a penalty method previously derived by Suh et al. [1]. In Part II [2], the mixed-penalty and velocity-pressure formulations are used to develop two-dimensional (2-D) quadrilateral and triangular elements and 3-D hexahedral and tetrahedral elements. Numerical examples, including those representative of soft tissue material testing and simple human joints, are used to validate the formulations and to illustrate their applications. A focus of this work is the comparison of alternate formulations for nonlinear problems. While it is demonstrated that both formulations produce a range of converging elements, the velocity-pressure formulation is found to be more efficient computationally.

Analysis of Cellular Organelles and Macromolecular Assemblies by Electron Energy Loss Spectroscopy

Recent advances in electron energy loss spectroscopy (EELS) have significantly extended the range of applications for biological microanalysis. For example, EELS can now detect physiological concentrations of the important element calcium in rapidly frozen cells with a sensitivity greater than that achievable by energy-dispersive x-ray spectroscopy (EDXS). It can also detect small numbers of phosphorus atoms bound to macromolecular assemblies, and measure water distributions in frozen hydrated tissue. Here we discuss some of these developments in the context of detection limits and mapping techniques in the scanning transmission electron microscope (STEM) and energy-filtering transmission electron microscope (EFTEM).The useful information about elemental composition in EELS of biological specimens generally resides in weak core-edge signals corresponding to atomic concentrations in the 10−5−10−3(1–100 mmol/kg dry weight) range. For example, the Ca L2,3signal/background ratio is typically only 10−3and it is necessary to measure differences in signal that are only 104 of the background. Changes in low-loss fine structure corresponding to varying chemical composition are also very subtle; for example, detection of a 3% change in water content requires reliable measurement of a 0.1 eV shift in the low-loss intensity maximum. To extract such information requires efficient parallel detection of the energy loss spectrum and a high-brightness source to provide a sufficient number of incident electrons. The dedicated STEM is particularly well-suited for analyzing low concentrations of biological elements. If desired, the probe current can be reduced into the picoampere range for low-dose, high-resolution imaging prior to elemental analysis. The STEM’S field-emission source can then be used to deliver a current approaching 10 nA into a ~10 nm diameter probe. High electron flux conditions are ideal for spectrum-imaging applications where adequate counting statistics must be achieved within a limited pixel dwell time. The cold field-emission source of the STEM has the additional advantage of providing electrons with a narrow energy spread of &lt;0.5 eV which is important in fine structure studies.

Changes in proteoglycan synthesis of chondrocytes in articular cartilage are associated with the time-dependent changes in their mechanical environment

Explant loading experiments were conducted to investigate the effect of load duration on proteoglycan synthesis. A compressive load of 0.1 MPa applied for 10 min was found to stimulate proteoglycan synthesis, while the same load applied for 20 h suppressed synthesis. This bimodal response suggests that the cells are responding to different mechanical stimuli as time progresses. A theoretical model has therefore been developed to describe the mechanical environment perceived by cells within soft hydrated tissues (e.g. articular cartilage) while the tissue is being loaded. The cells are modeled, using the biphasic theory, as fluid-solid inclusions embedded in and attached to a biphasic extracellular matrix of distinct material properties. A method of solution is developed which is valid for any axisymmetric loading configuration, provided that the cell radius, a, is small relative to the tissue height, h (i.e. h a ⪢ 1 ). A closed-form analytical solution for this inclusion problem is then presented for the confined compression configuration. Results from this model show that the mechanical environment in and around the cells is time dependent and inhomogeneous, and can be significantly influenced by differences in properties between the cell and the extracellular matrix.

Flows of Electrolytes Through Charged Hydrated Biological Tissue

In this paper, analyses of the flows of water and electrolytes through charged hydrated biologic tissues (e.g., articular cartilage) are presented. These analyses are based on the triphasic mechano-electrochemical theory developed by Lai and coworkers (1991). The problems analyzed are 1-D steady permeation flows generated by a hydraulic pressure difference and/or by an osmotic pressure difference across a finite thickness layer of the tissue. The theory allows for the complete determination of the ion concentration field, the matrix strain field as well as the ion and water velocity field inside the tissue during the steady permeation. For flows generated by a hydraulic pressure difference, the frictional drag induces a compaction of the solid matrix causing the fixed charge density (FCD) to increase and the neutral salt concentration to decrease in the downstream direction. Further, while both ions move downstream, but relative to the solvent (water), the anions (Cl−) move with the flow while the cations (Na+) move against the flow. The theory also predicts a well-known experimental finding that the apparent permeability decreases nonlinearly with FCD. For flows generated by an osmotic pressure difference, first, fluid flow varies with the FCD in a nonlinear and non-monotonic manner. Second, there exists a critical FCD below which negative osmosis takes place.

Low-temperature Scanning Electron Microscopy of Frozen Hydrated Apple Tissues and Surface Organisms

Frozen hydrated buds and epicarp of `Golden Delicious' apple (Malus domestica Borkh.) were observed with a low-temperature, field emission scanning electron microscope (SEM). In addition to observing surface features of these specimens, holders were modified to observe fractured specimens. A modified hinged holder retained both halves of a fractured specimen for examination of the complementary faces of frozen hydrated tissues. Low-temperature SEM avoided artifacts, such as extraction, solubilization, and shrinkage, which are normally encountered with chemical fixation, dehydration, and drying, respectively. The technique allowed observations of well-preserved frozen hydrated structures, such as the platelets of epicuticular wax; loosely associated organisms on plant surfaces, such as spider-mite eggs; delicate structures, such as fungal hyphae; and partially hydrated tissues, such as fruit epicarp and winter bud scales.