Viscoelastic properties of skin in Mov-13 and Tsk mice.

Viscoelastic properties of skin samples were measured in three types of mice (tight skin, Tsk, control and Mov-13), that are known to differ with regard to content of type I collagen. The experimental design used uniaxial stretching and measured the creep response and the complex compliance. The creep response was measured directly. The complex compliance was determined using a Wiener-Volterra constitutive model for each sample. The models were calculated from data obtained by applying a stress input having a pseudo-Gaussian waveform and measuring the strain response. The storage compliance of Mov-13 and control skin were similar and were greater than Tsk (p<0.001). The loss compliance of each group was significantly different (p<0.001) from each other group; Tsk had the lowest and control had the highest loss compliance. The phase angle of the Mov-13 and Tsk were similar and were less than the controls (p<0.001). The creep response was fit with a linear viscoelastic model. None of the parameters in the creep model differed between groups. The results indicate that gene-targeted and mutant animals have soft tissue mechanical phenotypes that differ in complex ways. Caution should be exercised when using such animals as models to explore the role of specific constituents on tissue properties.

Changes in ADC caused by tensile loading of rabbit achilles tendon: evidence for water transport.

Water diffusion measurements were performed on rabbit Achilles tendons during static tensile loading and tendons in an unloaded state. The apparent diffusion coefficient (ADC) was measured along two directions: parallel and perpendicular to the long axis of the tendon. Tendons were studied after being prepared in two ways: (a) after being stored frozen in phosphate-buffered saline (PBS) and (b) freshly isolated. Statistically significant directional anisotropy was observed in the ADC in all tendons. The ADC was significantly greater in the direction parallel to the long axis of the tendon than in the perpendicular direction. The anisotropy is attributed to the greater restrictions seen by the water molecules in the perpendicular direction and is consistent with the known geometry of the tendon. Storage in PBS caused tendons to swell. This increased the ADC measured along both directions and reduced the anisotropy. The existence of anisotropy in the ADC was not related to the orientation of the specimen in the magnet. The ADC increased along both directions following the application of a 5-N tensile load; the increase was greatest along the perpendicular axis of the tendon. In order to determine whether load-related changes in the ADC reflected changes in interfibrilar spacing, we used electron microscopy to measure load-related changes in fibril spacing. Load-related changes in fiber spacing could not account for the observed changes in the ADC. The increase in ADC caused by loading was attributed to the extrusion of tendon water into a bulk phase along the outside surface of the tendon. In PBS-stored samples, enough fluid was extruded that it could be visualized. The transient response of the ADC to a 5-N tensile load was also studied. The absolute ADC in both directions increased with loading and recovered to baseline upon unloading. The transient changes in ADC, for both loading and unloading, had a mean time constant of approximately 15 min. The magnitude of the load-induced transient ADC changes was comparable to that seen in the static-loading experiments.

Lipid deposition in rat aortas with intraluminal hemispherical plug stenosis. A morphological and biophysical study.

A new method was devised to create a stenosis in the rat abdominal aorta. To restrict blood flow, a hemispherical plug was inserted into the aorta through a renal artery. This type of intrinsic (intraluminal) stenosis minimizes possible intramural effects associated with external compression or ligation which severely deform the arterial wall. In the aorta of hypercholesterolemic rats, lipid deposits were distributed in crescent-shaped patches proximal and distal to the plug, whereas lipid deposition in the opposite aortic wall was inhibited. Based on enlarged physical scale models used to study the flow field, the regions of lipid deposition were found to coincide with regions of low shear stress, stagnation, and recirculation. Shear stress was elevated at the wall opposite the plug. These results show that when confounding mural effects are minimized, lipid deposition is promoted in regions of low shear stress with recirculation and inhibited in regions of elevated shear stress.

Variation of arterial compliance within the cardiac pressure pulse.

Past investigations of in vivo arterial behavior have concentrated on determining material properties based upon the maximum and minimum pressure and diameter measured over a pulse cycle. A new in vivo technique, based upon continuous measurement of pressure and flow, has been developed to study arterial compliance throughout the pulse cycle. Compliance in the abdominal aorta of rats showed different behavior during the rising and falling portion of the pressure pulse. Previous investigations of canine arteries which used different methods are consistent with these findings. This study demonstrates the utility of a new measurement technique and shows some trends in compliance within the pulse cycle which have neither been revealed by static tests nor by dynamic tests which focused on pulse averaged values.

A finite element based method to determine the properties of planar soft tissue.

A finite element based method to determine the incremental elastic material properties of planar membranes was developed and evaluated. The method is applicable to tissues that exhibit inhomogeneity, geometric and material nonlinearity, and anisotropy. Markers are placed on the tissue to form a four-node quadrilateral element. The specimen is loaded to an initial reference state, then three incremental loading sets are applied and the nodal displacements recorded. One of these loadings must include shear. These data are used to solve an over-determined system of equations for the tangent stiffness matrix. The method was first verified using analytical data. Next, data obtained from a latex rubber sheet were used to evaluate experimental procedures. Finally, experiments conducted on preconditioned rat skin revealed nonlinear orthotropic behavior. The vector norm comparing the applied and calculated nodal force vectors was used to evaluate the accuracy of the solutions.

The effect of diabetes on the viscoelastic properties of rat knee ligaments.

A series of experiments was performed to determine the effect of diabetes on the viscoelastic properties of knee joint ligaments. The experimental model was collateral ligaments from spontaneously diabetic, hyperglycemic (BBZDP/Wor) rats, and various controls including nondiabetic littermates, insulin treated diabetic rats, and alloxan treated rats. Material properties were measured using a dynamic, uniaxial loading paradigm. Ligaments were subjected to load controlled, sinusoidal tensile testing, using frequencies from 0.1 to 2.0 Hz. The resulting data were used to determine the storage and loss compliances of the ligaments. Storage compliance, which reflects tissue elastic properties, did not differ between groups. Loss compliance, which reflects the viscous component of the tissue response, was increased in the hyperglycemic animals. Thus, hyperglycemic diabetes affects tissue mechanical properties through the viscous rather than the elastic component of the response to dynamic loading. Rats treated with alloxan to induce diabetes did not show an increase in loss compliance.

Mechanical states encoded by stretch-sensitive neurons in feline joint capsule.

1. The sensitivity of group II joint afferents innervating cat knee joint capsule to in-plane stretch was studied in vitro. Single afferents were recorded from teased filaments of the posterior articular nerve. The capsule was stretched by applying forces through tabs along the edges of the capsule (3 tabs/edge) with the use of an apparatus that allowed for independent control of each load. The relationships between the neural responses of these afferents and the local continuum mechanical state of the joint capsule have been investigated. By appropriately loading the tissue margins, it was possible to establish states of uniaxial and biaxial tension, including shear. 2. Plane stress was calculated from the loads along the tissue margins. Stress at the location of the mechanoreceptor ending was estimated by interpolation. Strain was calculated from deformations of the capsule measured by tracking markers on its surface. Full characterization of tissue stress and strain made it possible to determine strain energy density and the magnitudes of other coordinate invariant mechanical quantities. 3. Individual afferents (n = 15) exhibited pronounced selectivity to the direction of applied stress and strain. There was no overall preferred orientation across neurons, and simple correlation of individual stress or strain components with the neuronal response revealed no consistent relationship between neuronal response and any single tensor component. However, linear multiple regression of the combined stress and strain components with the neuronal response revealed high correlation (mean R = 0.91), indicating that the measured mechanical states strongly determine the neuronal response. There was a much stronger relationship between neuronal response and stress variables than with strain variables. Simple correlation of the first invariant of the stress tensor with neuronal response had the highest mean correlation of the tensor quantities (R = 0.51). On average, strain energy density was only modestly correlated with the neural response (R = 0.28). 4. These findings indicate that capsule mechanoreceptors are encoding the local continuum mechanical state in the joint capsule. The neural response of these mechanoreceptors is more strongly correlated to local stress than to local strain.

Stretch-sensitive afferent neurons in cat knee joint capsule: sensitivity to axial and compression stresses and strains.

1. Experiments were performed to determine whether the response of stretch-sensitive mechanoreceptors to tissue deformation is caused by the axial stretching of the tissue or by the associated transverse compression of the tissue caused by the Poisson effect. 2. Single, stretch-sensitive mechanoreceptors were recorded in vitro in a preparation of innervated, isolated capsule from the cat knee. Afferents were isolated in a ligamentous capsule thickening that has a uniform geometry and parallel collagen fibers. The tissue was loaded axially while simultaneously stretching it around the surface of a cylinder to produce compression stresses and strains. Axial stresses and strains were measured or estimated. 3. By altering the diameter of the cylinder, given axial stresses and strains produced different levels of compression stresses and strains. It was possible to compare the neuronal response to pure uniaxial tension with the response when both axial stretching and transverse compression was applied. 4. In 8 of 11 experiments, transverse compression did not significantly change the response (P > 0.05). In one experiment, the response was decreased by compression. In the other two experiments, the response was increased but was not a function of the magnitude of the compressive stress. 5. Compressive strain was not significantly correlated with neuron response in any experiment. 6. The data do not sustain the model that the responses of stretch-sensitive neurons are due to local compression of the afferent ending.

Loading and deformation of the cat posterior knee joint capsule in axial and extension rotations.

Deformation and loading of the knee posterior joint capsule were studied in the cat, in extension rotations and in axial external (ER) and internal (IR) rotations at full extension. Loads were measured in the ligament along the upper edge of the capsule, using mechanically sensitive neurons that were calibrated as load cells. Strains were measured across the surface of the capsule, by tracking a set of markers attached to its surface. External rotations produced small loads in the cable: with applied moments of up to 0.25 Nm, cable tensions were less than 0.8 mPa. The cable was not loaded by internal rotations. Axial rotations produced predominantly shear strains in the capsule. Extension produced small loads in the ligament and the predominant capsule strain was tensile along the axis of the femur. These results show that the posterior capsule has a small role in resisting extension, a minimal role in providing axial stability in ER, and no such role in IR.

Mechanisms of hydroxyl free radical-induced cellular injury and calcium overloading in alveolar macrophages.

Excessive production of reactive oxygen radicals by alveolar macrophages is proposed to play an important role in oxidative lung injury. A major product oxygen radical formation is the highly reactive hydroxyl radical (.OH) generated via a biologic Fenton reaction. In addition to its known ability to induce lipid peroxidation, recent studies have suggested that the .OH may exert its cytotoxic effect through the alteration of [Ca2+]i homeostasis. To test this potential mechanism as well as to investigate the relationship between .OH and Ca2+ overloading in cytotoxic injury, isolated rat alveolar macrophages were exposed to externally generated radical system, H2O2 (0.01 to 1 mM) and Fe2+ (1 mM) and their [Ca2+]i levels and cell injury were monitored using quantitative fluorescence microscopy with the aid of the specific Ca2+ indicator, Fura-2, and membrane integrity indicator, propidium iodide. Electron spin resonance measurements using the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) confirmed the production of the .OH radical by this system. Upon the addition of the radicals, the macrophages displayed a rapid initial rise in [Ca2+]i which was followed by a slower but more pronounced [Ca2+]i elevation that reached a level 3 to 5 times higher than the basal level. This process preceded cell death as evident by nuclear propidium iodide fluorescence. Depletion of extracellular Ca2+ inhibited both the [Ca2+]i response and cell injury. Preincubation of the cells with the Ca2+ channel blocker verapamil or .OH radical scavenger mannitol similarly inhibited the [Ca2+]i rise and loss of viability. Firefly luciferase assay of cellular ATP content demonstrated that the alterations in [Ca2+]i following .OH treatment preceded the depletion of ATP.(ABSTRACT TRUNCATED AT 250 WORDS)

A composite micromechanical model for connective tissues: Part I--Theory.

A micromechanical model has been developed to study and predict the mechanical behavior of fibrous soft tissues. The model uses the theorems of least work and minimum potential energy to predict upper and lower bounds on material behavior based on the structure and properties of tissue components. The basic model consists of a composite of crimped collagen fibers embedded in an elastic glycosaminoglycan matrix. Upper and lower bound aggregation rules predict composite material behavior under the assumptions of uniform strain and uniform stress, respectively. Input parameters consist of the component material properties and the geometric configuration of the fibers. The model may be applied to a variety of connective tissue structures and is valuable in giving insight into material behavior and the nature of interactions between tissue components in various structures. Application of the model to rat tail tendon and cat knee joint capsule is described in a companion paper [2].

A composite micromechanical model for connective tissues: Part II--Application to rat tail tendon and joint capsule.

A micromechanical model of fibrous soft tissue has been developed which predicts upper and lower bounds on mechanical properties based on the structure and properties of tissue components by Ault and Hoffman [3, 4]. In this paper, two types of biological tissue are modeled and the results compared to experimental test data. The highly organized structure of rat tail tendon is modeled using the upper bound aggregation rule which predicts uniform strain behavior in the composite material. This model fits the experimental data and results in a correlation coefficient of 0.98. Applied to cat knee joint capsule, the lower bound aggregation rule of the model correlates with the data and predicts uniform stress within this more loosely organized tissue structure. These studies show that the nature of the interactions between the components in tissue differs depending upon its structure and that the biomechanical model is capable of analyzing such differences in structure.

Lipid deposition and intimal stress and strain. A study in rats with aortic stenosis.

These experiments were designed to study the topography of lipid deposition in the stenotic aorta of hypercholesterolemic rats, and to correlate it with flow conditions and intimal stresses and strains studied in a scale biophysical model and in a computer model. A 69% +/- 5% stenosis was produced with a U-shaped metal clip. One month to 8 months later, the aorta was studied en face by light microscopy after fixation and lipid staining. The intima in the throat of the stenosis was almost completely free of lipid, whereas symmetric lipid deposits occurred as bands just above and especially just below the stenosis; elsewhere lipid deposits appeared to be random. The flow data obtained from the scale model showed that the intima in the throat of the stenosis was subjected to an increase of as much as 20 times in shear stress, whereas the lipid deposits just above and just below the stenosis were associated with asymmetric flow conditions: the proximal area corresponded to a region of rapidly increasing shear stress, the distal area to a region of low to normal shear stress and separated flow. A finite element computer model based on the aortic deformations indicated that the endothelium at the inlet and outlet of the stenosis is subjected to a symmetric pattern of elevated stresses and strains. These results indicate that 1) the pattern of lipid deposition can not be adequately explained by a hypothesis based solely on flow conditions, and 2) lipid deposits can develop in areas of increased fluid shear stress, decreased fluid shear stress, and increased intimal strains.

Response of joint capsule neurons to axial stress and strain during dynamic loading in cat.

1. Experiments were conducted to test the hypothesis that the responses of joint capsule mechanoreceptors better encode tissue stress or tissue strain. The experimental model was a small ligament from the cat knee capsule, which was stretched uniaxially in vitro. Experiments were done with either force or displacement as the controlled variable, and with steps, sinusoids, or pseudorandom Gaussian noise (PGN) as the input function. 2. The strength of coupling between neural discharge and both strain and stress was quantified during step experiments using linear correlation coefficients. The correlation between the frequency of neural discharge and stress was 0.93 +/- 0.09 (SD). The correlation between frequency of neural discharge and strain was -0.91 +/- 0.06. The magnitudes of these correlation coefficients were not significantly different. 3. The strength of coupling between neural discharge and both strain and stress during sinusoidal and PGN experiments was quantified by the use of an information theoretic statistic, transinformation. Out of 282 sinusoidal runs, transinformation between neural discharge and stress was significantly greater than transinformation between strain and neural discharge 241 times. Transinformation between strain and neural discharge was significantly greater 15 times. 4. During PGN experiments, transinformation between stress and neural discharge was greater than transinformation between strain and neural discharge in all 19 experimental runs. 5. Conditional transinformation between strain and neural discharge, given stress, was calculated for all sinusoidal and pseudorandom experiments. This statistic was greater than zero in 268 out of 289 experimental runs, indicating that a component of strain independent of stress is being signaled in the neural discharge.

Calibrating joint capsule mechanoreceptors as in vivo soft tissue load cells.

A method has been developed whereby the discharge of mechanically sensitive neurons from the cat knee joint capsule can be calibrated and used as load cells. The neurons are located in the upper edge of the capsule which has been previously modeled as a suspension cable and where the loading has been shown to be one dimensional. The calibration procedure relies upon applying known point loads to the cable and measuring its shape. The biomechanical model is then used to compute the cable tension at the neuron location. Results for 20 neurons showed a strong linear relationship between the tension and the frequency of neuronal discharge (r = 0.96, S.D. = 0.05). For 11 of these neurons the in vivo calibration was verified by subsequently excising the posterior capsule and recording from the same neuron while subjecting the cable to measured uniaxial loads. Results showed good agreement between the in vivo and in vitro calibrations. Once calibrated these neurons can be used as load sensors to study in vivo joint loading.

Measurement of joint capsule tissue loading in the cat knee using calibrated mechanoreceptors.

The vertical loading in the posterior capsule of the cat knee has been measured while the knee is rotated into hyperextension. Tissue loading was determined using a previously verified model of the capsule that represents its upper edge as a catenary suspension cable. Tensile loads in the cable were measured using the discharge of mechanoreceptive sensory neurons that had been calibrated as load sensors. The results revealed that the capsule is very lightly loaded in extension rotations. Less than 4% of the applied moment is sustained by the capsule.

Endothelial adaptations in aortic stenosis. Correlation with flow parameters.

A 69 +/- 5% stenosis was produced in the rat aorta, with the purpose of correlating endothelial changes with local flow patterns and with levels of shear stress; the hydrodynamic data were obtained from a scaled-up model of the stenosed aorta. In the throat of the stenosis, where shear stress values were 15-25 times normal, the endothelium was stripped off within 1 hour. It regenerated at half the rate of controls but modulated into a cell type that could withstand the increased shear stress. Adaptations included changes in cell orientation, number, length, width, thickness, stress fibers, and anchoring structures, as well as changes in the length, argyrophilia, and permeability of the junctions. Areas of either elongated or "polygonal" cells consistently developed at the same sites in relation to the stenosis, but the hydrodynamic data showed that they did not always correspond (as had been anticipated) to high and low shear, respectively. It is concluded that endothelial cell shape in the living artery must be determined by some other factor(s) in addition to shear stress.

A biomechanical model of the posterior knee capsule of the cat.

A biomechanical model is presented which represents the upper edge of the posterior knee capsule in the cat as a two-segment, vertically loaded catenary suspension cable from which the capsule sheet is suspended. Data are presented which show that the upper edge of the capsule is organized as a cable, which spans the notch between the femoral condyles. When a point load is applied to the cable, measurement of the cable shape allows for calculation of the cable tension and the downward distributed loads acting on the cable. This method was used to measure the in-vivo cable tension and the distributed downward loading acting on the capsule cable. The results show that the lateral side of the posterior joint capsule sustains a higher loading than the medial side.

A method for measuring strains in soft tissue.

A finite element based method has been developed for measuring strains in soft tissue. An array of markers is placed on the tissue surface and treated as nodes of a four node isoparametric element. The displacements of the marker centroids are directly measured using a high sensitivity television camera. Finite element method mathematics are then used to calculate the plane strain tensor at any point inside the element. The method has been implemented using non-rectangular elements that are approximately 2 mm on each side.

Ruffini mechanoreceptors in isolated joint capsule: responses correlated with strain energy density.

Mechanoreceptive afferents innervating the posterior capsule of the cat knee joint were recorded in a preparation of isolated capsule. The purpose of the experiments was to identify mechanical states in the capsule that were associated with afferent discharge. The capsule was excised from the knee with its bone attachments intact, so that the geometry of the capsule could be reproduced in vitro. The capsule was deformed, and measurements were made of stresses and strains in the plane of the capsule. Afferent discharge was correlated with each of the components of plane stress, plane strain, and strain energy density (SED). SED, the stored elastic energy at the receptor location, was the only mechanical variable that was consistently positively correlated with afferent discharge. A model of the Ruffini-type receptor is presented that accounts for the sensitivity to SED.