Biomechanical properties of human articular cartilage under compressive loads

The function of articular cartilage is to support and distribute loads and to provide lubrication in the diarthrodial joints. Cartilage function is described by proper mechanical and rheological properties, strain and depth-dependent, which are not completely assessed. Unconfined and confined compression are commonly used to evaluate the Young's modulus (E) and the aggregate modulus (H(A)), respectively. The Poisson's ratio (nu) can be calculated indirectly from the equilibrium compression data, or using the biphasic indentation technique; it has recently been optically evaluated by using video microscopy during unconfined compression. The transient response of articular cartilage during confined compression depends on its permeability k; a constant value of k can be easily identified by a simple analytical model of confined compression tests, whereas more complex models or direct measurements (permeation tests) are needed to study the permeability dependence on deformation. A poroelastic finite element model of articular cartilage was developed for this purpose. The elastic parameters (E,nu) of the model were evaluated performing unconfined compression creep tests on human articular cartilage disks, whereas k was identified from the confined test response. Our combined experimental and computational method can be used to identify the parameters that define the permeability dependence on deformation, as a function of depth from articular surface.     

The strain-dependent osmotic pressure and stiffness of the bovine nucleus pulposus apportioned into ionic and non-ionic contributors

Elucidation of the load-bearing mechanism of the nucleus pulposus (NP) facilitates understanding of the mechanical and metabolic functioning of the intervertebral disc and provides key data for mathematical models. Negatively charged proteoglycans in the NP generate an ionic osmotic pressure, pi(i), which contributes to the tissue's resistance to load and, moreover, is the main mechanism by which the unloaded disc rehydrates. Functionally important, pi(i) has seldom been investigated in situ and, crucially, its variation with strain has not been reported. In a confined compression apparatus, we aimed to apportion the strain-dependent load-bearing mechanism of the NP at equilibrium to the tissue matrix and ionic osmotic pressure; and to determine whether any proteoglycan loss occurs during confined compression testing. Forty-eight confined compression experiments were conducted in isotonic (0.15M NaCl) and hypertonic (3.0 and 6.1M NaCl) external solutions in single and multiple step-strain protocols. The 6.1M NaCl external solution was needed to eliminate as much of the ionic effects as possible. The ionic osmotic pressure was well described by pi(i)=19.1lambda(-1.58) (R(2)=0.992), and was approximately 70% of the applied load at equilibrium, independent of lambda. The effective aggregate modulus, H(A)(eff), also increased with strain: H(A)(eff)=59.0lambda(-2.18). Concentrations of sulphated glycosaminoglycans were obtained for the samples tested in isotonic NaCl with no proteoglycan loss detected from the confined compression tests. These results highlight the non-linearity of the stress-strain response of NP tissue and the necessity to include a non-linear function for osmotic pressure in mathematical models of this tissue.     

A linear laser scanner to measure cross-sectional shape and area of biological specimens during mechanical testing

Measure of the cross-sectional area (CSA) of biological specimens is a primary concern for many biomechanical tests. Different procedures are presented in literature but besides the fact that noncontact techniques are required during mechanical testing, most of these procedures lack accuracy or speed. Moreover, they often require a precise positioning of the specimen, which is not always feasible, and do not enable the measure of the same section during tension. The objective of this study was to design a noncontact, fast, and accurate device capable of acquiring CSA of specimens mounted on a testing machine. A system based on the horizontal linear displacement of two charge-coupled device reflectance laser devices next to the specimen, one for each side, was chosen. The whole measuring block is mounted on a vertical linear guide to allow following the measured zone during sample tension (or compression). The device was validated by measuring the CSA of metallic rods machined with geometrical shapes (circular, hexagonal, semicircular, and triangular) as well as an equine superficial digital flexor tendon (SDFT) in static condition. We also performed measurements during mechanical testing of three SDFTs, obtaining the CSA variations until tendon rupture. The system was revealed to be very fast with acquisition times in the order of 0.1 s and interacquisition time of about 1.5 s. Measurements of the geometrical shapes yielded mean errors lower than 1.4% (n=20 for each shape) while the tendon CSA at rest was 90.29 ± 1.69 mm(2) (n=20). As for the tendons that underwent tension, a mean of 60 measures were performed for each test, which lasted about 2 min until rupture (at 20 mm/min), finding CSA variations linear with stress (R(2)>0.85). The proposed device was revealed to be accurate and repeatable. It is easy to assemble and operate and capable of moving to follow a defined zone on the specimen during testing. The system does not need precise centering of the sample and can perform noncontact measures during mechanical testing; therefore, it can be used to measure variations of the specimen CSA during a tension (or compression) test in order to determine, for instance, the true stress and transverse deformations.     

Confined and unconfined stress relaxation of cartilage: appropriateness of a transversely isotropic analysis.

Previous studies have shown that stress relaxation behavior of calf ulnar growth plate and chondroepiphysis cartilage can be described by a linear transverse isotropic biphasic model. The model provides a good fit to the observed unconfined compression transients when the out-of-plane Poisson's ratio is set to zero. This assumption is based on the observation that the equilibrium stress in the axial direction (deltaz) is the same in confined and unconfined compression, which implies that the radial stress deltar = 0 in confined compression. In our study, we further investigated the ability of the transversely isotropic model to describe confined and unconfined stress relaxation behavior of calf cartilage. A series of confined and unconfined stress relaxation tests were performed on calf articular cartilage (4.5 mm diameter, approximately 3.3 mm height) in a displacement-controlled compression apparatus capable of measuring delta(z) and delta(r). In equilibrium, delta(r) > 0 and delta(z) in confined compression was greater than in unconfined compression. Transient data at each strain were fitted by the linear transversely isotropic biphasic model and the material parameters were estimated. Although the model could provide good fits to the unconfined transients, the estimated parameters overpredicted the measured delta(r). Conversely, if the model was constrained to match equilibrium delta(r), the fits were poor. These findings suggest that the linear transversely isotropic biphasic model could not simultaneously describe the observed stress relaxation and equilibrium behavior of calf cartilage.     

A Near-field Nanosource Based on Surface Plasmon Bragg Reflectors and Nanocavity

We demonstrate a type of confined nanosource based on surface plasmon band-gap structure consisting of a nanocavity surrounded by grooves. A single, localized, and non-radiating central peak is obtained and can be used as a nanosource. The characteristics of the surface plasmon polariton (SPP) field in the vicinity of the structures with different geometrical parameters are investigated experimentally. A confined central peak is obtained in the nanocavity. The full width at half maximum of the central peak is beyond the diffraction limit and changes little during 600 nm distance away from the sample surface. With the modifications of the geometrical parameters, the central peak intensity can be enhanced and the sidelobes can be suppressed. The physical origin of the enhancement and the surface-sensitivity is explored theoretically. These phenomena demonstrate the abilities of the structures to collect the electromagnetic field and to tailor the SPP field profile. This type of SPP-based nanosource is promising to be applied in near-field imaging, data storage, optical manipulation, and localized spectrum excitation, and has potential applications in nano-photonics devices based on SPPs.     

Impacted morselized cancellous bone: mechanical effects of defatting and augmentation with fine hydroxyapatite particles

Geotechnical engineering testing techniques were used to study the mechanical properties of morselized cancellous bone (MCB) and the effects of defatting and augmentation with fine hydroxyapatite (HA) particles. Bovine and human cancellous bone was morselized, rinsed, and manually squeezed to remove excess fluid, producing a standard surgical MCB sample that was also used as a control. Some of the MCB was defatted with heat and detergent and mixed with HA particles in ratios ranging from 0% to 100% HA. Compaction tests were used to determine the effects of moisture content and the amount of MCB that can be packed into a confined space. One-dimensional consolidation tests were used to determine the uniaxial strain behavior, confined modulus, and steady-state creep rate. The compaction tests demonstrated that defatting and adding HA particles significantly increased density. The one-dimensional consolidation tests showed that strain was decreased, modulus was increased and the creep rate was decreased by defatting and adding HA.     

Comparison of the equilibrium response of articular cartilage in unconfined compression,confined compression and indentation

At mechanical equilibrium, articular cartilage is usually characterized as an isotropic elastic material with no interstitial fluid flow. In this study, the equilibrium properties (Young's modulus, aggregate modulus and Poisson's ratio) of bovine humeral, patellar and femoral cartilage specimens (n=26) were investigated using unconfined compression, confined compression, and indentation tests. Optical measurements of the Poisson's ratio of cartilage were also carried out. Mean values of the Young's modulus (assessed from the unconfined compression test) were 0.80+/-0.33, 0.57+/-0.17 and 0.31+/-0.18MPa and of the Poisson's ratio (assessed from the optical test) 0.15+/-0.06, 0.16+/-0.05 and 0.21+/-0.05 for humeral, patellar, and femoral cartilages, respectively. The indentation tests showed 30-79% (p<0.01) higher Young's modulus values than the unconfined compression tests. In indentation, values of the Young's modulus were independent of the indenter diameter only in the humeral cartilage. The mean values of the Poisson's ratio, obtained indirectly using the mathematical relation between the Young's modulus and the aggregate modulus in isotropic material, were 0.16+/-0.06, 0.21+/-0.05, and 0.26+/-0.08 for humeral, patellar, and femoral cartilages, respectively. We conclude that the values of the elastic parameters of the cartilage are dependent on the measurement technique in use. Based on the similar values of Poisson's ratios, as determined directly or indirectly, the equilibrium response of articular cartilage under unconfined and confined compression is satisfactorily described by the isotropic elastic model. However, values of the isotropic Young's modulus obtained from the in situ indentation tests are higher than those obtained from the in vitro unconfined or confined compression tests and may depend on the indenter size in use.     

Confined compression experiments on bovine nucleus pulposus and annulus fibrosus: sensitivity of the experiment in the determination of compressive modulus and hydraulic permeability

The biphasic material properties for nucleus pulposus tissue in confined compression have not been reported previously, and are required for a better understanding of intervertebral disc function and to provide material properties for use in finite-element models. The aims of this study were to determine linear and non-linear material properties for nucleus pulposus and annulus fibrosus tissues in confined compression, to define the influence of swelling conditions on these properties, and to determine the changes in the compressive modulus and hydraulic permeability induced by the repetition of the stress-relaxation experiment after a return to swelling pressure equilibrium. Specimens from caudal bovine nucleus and annulus were tested in confined compression stress-relaxation experiments and analyzed to quantify the compressive modulus and hydraulic permeability using linear and non-linear biphasic models. Our results suggested the use of confined swelling pre-test condition and non-linear biphasic model, which provided the material parameters with lowest relative variance and water content most representative of physiological conditions. Smaller compressive modulus and higher hydraulic permeability were obtained for the nucleus (H(A0)=0.31+/-0.04 MPa, k(0)=0.67+/-0.09 x 10(-15)m(4)/Ns) than for the annulus (H(A0)=0.74+/-0.13 MPa, k(0)=0.23+/-0.19 x 10(-15)m(4)/Ns), with relatively weak non-linearities. Strains up to 20% resulted in material properties that were significantly altered upon retesting. These altered material properties are an effort to quantify non-recoverable damage that occurs in disc tissue and suggest that in vivo exposure of disc tissues to low strain-rate and high-deformation loading conditions which outpace biological repair may result in altered mechanical behaviors.     

Viscoelastic properties of ovine adipose tissue covering the gluteus muscles

Pressure-related deep tissue injury (DTI) is a life-risking form of pressure ulcers threatening immobilized and neurologically impaired patients. In DTI, necrosis of muscle and enveloping adipose tissues occurs under intact skin, owing to prolonged compression by bony prominences. Modeling the process of DTI in the buttocks requires knowledge on viscoelastic mechanical properties of the white adipose tissue covering the gluteus muscles. However, this information is missing in the literature. Our major objectives in this study were therefore to (i) measure short-term (H(S)) and long-term (H(L)) aggregate moduli of adipose tissue covering the glutei of sheep, (ii) determine the effects of preconditioning on H(S) and H(L), and (iii) determine the time course of stress relaxation in terms of the transient aggregate modulus H(t) in nonpreconditioned (NPC) and preconditioned (PC) tissues. We tested 20 fresh tissue specimens (from 20 mature animals) in vitro: 10 specimens in confined compression for obtaining the complete H(t) response to a ramp-and-hold protocol (ramp rate of 300 mms), and 10 other specimens in swift indentations for obtaining comparable short-term elastic moduli at higher ramp rates (2000 mms). We found that H(S) in confined compression were 28.9+/-14.9 kPa and 18.1+/-6.9 kPa for the NPC and PC specimens, respectively. The H(L) property, 10.3+/-4.2 kPa, was not affected by preconditioning. The transient aggregate modulus H(t) always reached the plateau phase (less than 10% difference between H(t) and H(L)) within 2 min, which is substantially shorter than the times for DTI onset reported in previous animal studies. The short-term elastic moduli at high indentation rates were 22.6+/-10 kPa and 15.8+/-9.4 kPa for the NPC and PC test conditions, respectively. Given a Poisson's ratio of 0.495, comparison of short-term elastic moduli between the high and slow rate tests indicated a strong deformation-rate dependency. The most relevant property for modeling adipose tissue as related to DTI is found to be H(L), which is conveniently unaffected by preconditioning. The mechanical characteristics of white adipose tissue provided herein are useful for analytical as well as numerical models of DTI, which are essential for understanding this serious malady.     

Transverse elasticity and blood perfusion of sciatic nerves under in situ circular compression

Biomechanical properties and microcirculation of peripheral nerves under circular compression are vital factors for nerve repair and for developing neural prostheses. Quasi-static circular compression experiments on six rabbit sciatic nerves were performed. The mean estimated Young's modulus of the sciatic nerves in the transverse direction was 66.9+/-8.0 kPa. The blood perfusion of the nerve started to decrease at a mean pressure of 30.5 mmHg and reached a stable lower level of 30% of pre-compression value at 102.8 mmHg. The findings may make a contribution to safer design of cuff electrodes to be used in neural prostheses.     

Depth- and strain-dependent mechanical and electromechanical properties of full-thickness bovine articular cartilage in confined compression.

Compression tests have often been performed to assess the biomechanical properties of full-thickness articular cartilage. We tested whether the apparent homogeneous strain-dependent properties, deduced from such tests, reflect both strain- and depth-dependent material properties. Full-thickness bovine articular cartilage was tested by oscillatory confined compression superimposed on a static offset up to 45%. and the data fit to estimate modulus, permeability, and electrokinetic coefficient assuming homogeneity. Additional tests on partial-thickness cartilage were then performed to assess depth- and strain-dependent properties in an inhomogeneous model, assuming three discrete layers (i = 1 starting from the articular surface, to i = 3 up to the subchondral bone). Estimates of the zero-strain equilibrium confined compression modulus (H(A0)), the zero-strain permeability (kp0) and deformation dependence constant (M), and the deformation-dependent electrokinetic coefficient (ke) differed among individual layers of cartilage and full-thickness cartilage. HiA0 increased from layer 1 to 3 (0.27 to 0.71 MPa), and bracketed the apparent homogeneous value (0.47 MPa). ki(p0) decreased from layer 1 to 3 (4.6 x 10(-15) to 0.50 x 10(-15) m2/Pa s) and was less than the homogeneous value (7.3 x 10(-15) m2/Pa s), while Mi increased from layer 1 to 3 (5.5 to 7.4) and became similar to the homogeneous value (8.4). The amplitude of ki(e) increased markedly with compressive strain, as did the homogeneous value: at low strain, it was lowest near the articular surface and increased to a peak in the middle-deep region. These results help to interpret the biomechanical assessment of full-thickness articular cartilage.     

Human tolerance to heat strain during exercise: influence of hydration.

This study determined whether 1) exhaustion from heat strain occurs at the same body temperatures during exercise in the heat when subjects are euhydrated as when they are hypohydrated, 2) aerobic fitness influences the body temperature at which exhaustion from heat strain occurs, and 3) curves could be developed to estimate exhaustion rates at a given level of physiological strain. Seventeen heat-acclimated men [maximal oxygen uptake (VO2max) from 45 to 65 ml.kg-1.min-1] attempted two heat stress tests (HSTs): one when euhydrated and one when hypohydrated by 8% of total body water. The HSTs consisted of 180 min of rest and treadmill walking (45% VO2max) in a hot-dry (ambient temperature 49 degrees C, relative humidity 20%) environment. The required evaporative cooling (Ereq) exceeded the maximal evaporative cooling capacity of the environment (Emax); thus thermal equilibrium could not be achieved and 27 of 34 HSTs ended by exhaustion from heat strain. Our findings concerning exhaustion from heat strain are 1) hypohydration reduced the core temperature that could be tolerated; 2) aerobic fitness, per se, did not influence the magnitude of heat strain that could be tolerated; 3) curves can be developed to estimate exhaustion rates for a given level of physiological strain; and 4) exhaustion was rarely associated with a core temperature up to 38 degrees C, and it always occurred before a temperature of 40 degrees C was achieved. These findings are applicable to heat-acclimated individuals performing moderate-intensity exercise under conditions where Ereq approximates or exceeds Emax and who have high skin temperatures.     

Experimental verification of the roles of intrinsic matrix viscoelasticity and tension-compression nonlinearity in the biphasic response of cartilage

A biphasic-CLE-QLV model proposed in our recent study [2001, J. Biomech. Eng., 123, pp. 410-417] extended the biphasic theory of Mow et al. [1980, J. Biomech. Eng., 102, pp. 73-84] to include both tension-compression nonlinearity and intrinsic viscoelasticity of the cartilage solid matrix by incorporating it with the conewise linear elasticity (CLE) model [1995, J. Elasticity, 37, pp. 1-38] and the quasi-linear viscoelasticity (QLV) model [Biomechanics: Its foundations and objectives, Prentice Hall, Englewood Cliffs, 1972]. This model demonstrates that a simultaneous prediction of compression and tension experiments of articular cartilage, under stress-relaxation and dynamic loading, can be achieved when properly taking into account both flow-dependent and flow-independent viscoelastic effects, as well as tension-compression nonlinearity. The objective of this study is to directly test this biphasic-CLE-QLV model against experimental data from unconfined compression stress-relaxation tests at slow and fast strain rates as well as dynamic loading. Twelve full-thickness cartilage cylindrical plugs were harvested from six bovine glenohumeral joints and multiple confined and unconfined compression stress-relaxation tests were performed on each specimen. The material properties of specimens were determined by curve-fitting the experimental results from the confined and unconfined compression stress relaxation tests. The findings of this study demonstrate that the biphasic-CLE-QLV model is able to describe the strain-rate-dependent mechanical behaviors of articular cartilage in unconfined compression as attested by good agreements between experimental and theoretical curvefits (r2 = 0.966 +/- 0.032 for testing at slow strain rate; r2 = 0.998 +/- 0.002 for testing at fast strain rate) and predictions of the dynamic response (r2 = 0.91 +/- 0.06). This experimental study also provides supporting evidence for the hypothesis that both tension-compression nonlinearity and intrinsic viscoelasticity of the solid matrix of cartilage are necessary for modeling the transient and equilibrium responses of this tissue in tension and compression. Furthermore, the biphasic-CLE-QLV model can produce better predictions of the dynamic modulus of cartilage in unconfined dynamic compression than the biphasic-CLE and biphasic poroviscoelastic models, indicating that intrinsic viscoelasticity and tension-compression nonlinearity of articular cartilage may play important roles in the load-support mechanism of cartilage under physiologic loading.     

Hydration of ds-DNA and ss-DNA by neutron quasielastic scattering

Quasielastic neutron scattering measurements were performed in hydrated samples of ds-DNA and ss-DNA. The samples were hydrated in a high relative humidity atmosphere, and their final water content was 0.559 g H(2)O/g ds-DNA and 0.434 g H(2)O/g ss-DNA. The measurements were performed at 8 and 5.2 A for the ds-DNA sample, and at 5.2 A for the ss-DNA sample. The temperature was in both cases 298 K. Analysis of the obtained data indicates that in the ds-DNA sample we can distinguish two types of protons-those belonging to water molecules strongly attached to the ds-DNA surface and another fraction belonging to water that diffuses isotropically in a sphere of radius 2.8 A, with a local diffusion coefficient of 2.2 x 10(-5) cm(2) s(-1). For ss-DNA, on the other hand, no indication was found of motionally restricted or confined water. Further, the fraction of protons strongly attached to the ds-DNA surface corresponds to 0.16 g H(2)O/g ds-DNA, which equals the amount of water that is released by ds-DNA upon thermal denaturation, as studied by one of us (G.M.) by differential scanning calorimetry. This value also equals the difference between the critical hydration values of ds-DNA and ss-DNA, also determined by DSC. These results represent, thus, a completely independent measurement of water characteristics and behavior in ds- and ss-DNA at critical hydration values, and therefore substantiate the previous suggestions/conclusions of the results obtained by calorimetry.     

Kinetic characteristics of 22Na transport in human and rat erythrocytes during cytoplasm acidification and cell compression

Under normal conditions (pH0 = 7.4, pHi = 7.1-7.2) amiloride, a Na+/H+ exchange inhibitor, does not influence Na+ intake by human and rat erythrocytes. Acidification of the cytoplasm (pHi approximately 6.4) is accompanied by the acceleration of 22Na intake, which is decreased after addition of 1 mM amiloride (by 50 and 80%, respectively). The Ki value of amiloride for human and rat erythrocytes is 30 and 250 microM, respectively. In rat erythrocytes the dependence of the rate of the delta pH-induced incorporation of 22Na on Na+ concentration is described by a saturation curve (K0.5 for Na0+ is approximately 40 mM), whereas in human erythrocytes it obeys the diffusion kinetics. These results suggest that the Na+/H+ exchange takes place in rat erythrocytes, but is absent in human erythrocytes. In rat erythrocytes the Na+/H+ exchange can be induced by cell compression which can be caused either by decreasing the KCl content (after addition of valinomycin) or by increasing the osmolarity of the medium (in the presence of sucrose). The rate of Na+/H+ exchange induced by cell compression is increased by 60-70% after addition of protein kinases A and C activators. No effect of intracellular Ca2+ on the rate of the Na+/H+ exchange in rat erythrocytes is observed.     

In situ measurement of articular cartilage deformation in intact femoropatellar joints under static loading

The deformational behavior of articular cartilage has been investigated in confined and unconfined compression experiments and indentation tests, but to date there exist no reliable data on the in situ deformation of the cartilage during static loading. The objective of the current study was to perform a systematic study into cartilage compression of intact human femoro-patellar joints under short- and long-term static loading with MR imaging. A non-metallic pneumatic pressure device was used to apply loads of 150% body weight to six joints within the extremity coil of an MRI scanner. The cartilage was delineated during the compression experiment with previously validated 2D and 3D fat-suppressed gradient echo sequences. We observed a mean (maximal) in situ deformation of 44% (57%) in patellar cartilage after 32 h of loading (mean contact pressure 3.6 MPa), the femoral cartilage showing a smaller amount of deformation than the patella. However, only around 7% of the final deformation (3% absolute deformation) occurred during the first minute of loading. A 43% fluid loss from the interstitial patellar matrix was recorded, the initial fluid flux being 0.217 +/- 0.083 microm/s, and a high inter-individual variability of the deformational behavior (coefficients of variation 11-38%). In conjunction with finite-element analyses, these data may be used to compute the load partitioning between the solid matrix and fluid phase, and to elucidate the etiologic factors relevant in mechanically induced osteoarthritis. They can also provide direct estimates of the mechanical strain to be encountered by cartilage transplants.     

A bioinformatic filter for improved base-call accuracy and polymorphism detection using the Affymetrix GeneChip whole-genome resequencing platform

DNA resequencing arrays enable rapid acquisition of high-quality sequence data. This technology represents a promising platform for rapid high-resolution genotyping of microorganisms. Traditional array-based resequencing methods have relied on the use of specific PCR-amplified fragments from the query samples as hybridization targets. While this specificity in the target DNA population reduces the potential for artifacts caused by cross-hybridization, the subsampling of the query genome limits the sequence coverage that can be obtained and therefore reduces the technique's resolution as a genotyping method. We have developed and validated an Affymetrix Inc. GeneChip(R) array-based, whole-genome resequencing platform for Francisella tularensis, the causative agent of tularemia. A set of bioinformatic filters that targeted systematic base-calling errors caused by cross-hybridization between the whole-genome sample and the array probes and by deletions in the sample DNA relative to the chip reference sequence were developed. Our approach eliminated 91% of the false-positive single-nucleotide polymorphism calls identified in the SCHU S4 query sample, at the cost of 10.7% of the true positives, yielding a total base-calling accuracy of 99.992%.     

Effect of non-uniform thickness of samples in stress relaxation tests under unconfined compression of samples of articular discs

A precise information of the biomechanical properties of soft tissues is required to develop a suitable simulation model, with which the distribution of stress and strain in the complex structures can be estimated. Many soft tissues have been mechanically characterized by stress relaxation tests under unconfined or confined compression. In general, full-thickness samples are extracted to reduce the damage in the tissue as much as possible. However, it is not guaranteed that these samples have a uniform thickness or, in other words, planar parallel faces. In particular, in the articular disc of the temporomandibular joint, many studies can be found testing full-thickness samples for which that thickness is known to be non-uniform, while making the assumption of uniaxial stress state to extract the mechanical properties from those tests. That inaccuracy may have a strong influence in some cases and needs a profound revision. The main goal of this work is to quantify the error committed in that assumption and the influence of the variation of thickness on that error in a particular test: stress relaxation tests under unconfined compression. Based on this error and defining an allowable tolerance, a criterion is established to reject samples depending on their aspect ratio.     

Substitution of Azotobacter vinelandii hydrogenase small-subunit cysteines by serines can create insensitivity to inhibition by O2 and preferentially damages H2 oxidation over H2 evolution.

Mutants in which conserved cysteines 294, 297 or 64 and 65 of the Azotobacter vinelandii hydrogenase small subunit were replaced by serines were studied. Cysteines 294 and 297 are homologous to cysteines 246 and 249 of the Desulfovibrio gigas hydrogenase, and these cysteines are ligands to the [3Fe-4S] clusters (A. Volbeda, M.-H. Charon, C. Piras, E. C. Hatchikian, M. Frey, and J. C. Fontecilla-Camps, Nature (London) 373:580-587, 1995). Cysteine 65 is homologous to cysteine 20 of the D. gigas hydrogenase, and this cysteine is a ligand to the proximal [4Fe-4S] cluster. All three mutants retained some hydrogenase activity. All three mutants studied had H2 oxidation-to-H2 evolution activity ratios with whole cells of approximately 1.5, compared with 46 for the wild type. The changes preferentially deplete H2 oxidation activity, while having less effect on evolution. The K64,65C-->S hydrogenase was partially purified and had a specific activity for the evolution reaction that was 22% that of the wild type, while the oxidation-specific activity was 2% that of the wild type. Because cysteine 65 provides a ligand to the proximal [4Fe-4S] cluster, this cluster can be altered without entirely eliminating enzyme activity. Likewise, the detection of H2 evolution and H2 oxidation activities with whole cells and membranes of the K294C-->S and K297C-->S mutants indicates that the [3Fe-4S] cluster can also be altered or possibly eliminated without entirely eliminating enzyme activity. Membranes with K294C-->S or K297C-->S hydrogenase were uninhibited by O2 in H2 oxidation and uninhibited by H2 in H2 evolution. Wild-type membranes and membranes with K64,65C-->S hydrogenase were both sensitive to these inhibitors. These data indicate that the [3Fe-4S] cluster controls the reversible inhibition of hydrogenase activity by O2 or H2.