In vivo and in vitro correlation of trachealis muscle contraction in dogs.

Maximal trachealis muscle shortening in vivo was compared with that in vitro in seven anesthetized dogs. In addition, the effect of graded elastic loads on the muscle was evaluated in vitro. In vivo trachealis muscle shortening, as measured using sonomicrometry, revealed maximal active shortening to be 28.8 +/- 11.7% (SD) of initial length. Trachealis muscle preparations from the same animals were studied in vitro to evaluate isometric force generation, isotonic shortening, and the effect of applying linear elastic loads to the trachealis muscle during contraction from optimal length. Maximal isotonic shortening was 66.8 +/- 8.4% of optimal length in vitro. Increasing elastic loads decreased active shortening and velocity of shortening in vitro in a hyperbolic manner. The elastic load required to decrease in vitro shortening to the extent of the shortening observed in vivo was similar to the estimated load provided by the tracheal cartilage. We conclude that decreased active shortening in vivo is primarily due to the elastic afterload provided by cartilage.     

In vivo liver tissue mechanical properties by Transient Elastography: comparison with Dynamic Mechanical Analysis

Understanding the mechanical properties of human liver is one of the most critical aspects of its numerical modeling for medical applications or impact biomechanics. Generally, model constitutive laws come from in vitro data. However, the elastic properties of liver may change significantly after death and with time. Furthermore, in vitro liver elastic properties reported in the literature have often not been compared quantitatively with in vivo liver mechanical properties on the same organ. In this study, both steps are investigated on porcine liver. The elastic property of the porcine liver, given by the shear modulus G, was measured by both Transient Elastography (TE) and Dynamic Mechanical Analysis (DMA). Shear modulus measurements were realized on in vivo and in vitro liver to compare the TE and DMA methods and to study the influence of testing conditions on the liver viscoelastic properties. In vitro results show that elastic properties obtained by TE and DMA are in agreement. Liver tissue in the frequency range from 0.1 to 4 Hz can be modeled by a two-mode relaxation model. Furthermore, results show that the liver is homogeneous, isotropic and more elastic than viscous. Finally, it is shown in this study that viscoelastic properties obtained by TE and DMA change significantly with post mortem time and with the boundary conditions.     

Effects of elastic loading on porcine trachealis muscle mechanics

To shorten in vivo, airway smooth muscle must overcome an elastic load provided by cartilage and lung parenchyma. We examined the effects of linear elastic loads (0.2-80 g/cm) on the active changes in porcine trachealis muscle length and tension in response to electrical field stimulation in vitro. Increasing elastic loads produced an exponential decrease in the shortening and velocity of shortening while causing an increase in tension generation of muscle strips stimulated by electrical field stimulation. Shortening was decreased by 50% at a load of 8 g/cm. At small elastic loads (less than or equal to 1 g/cm) contractile responses approximated isotonic responses (shortening approximately 60% of starting length), whereas at large loads (20 g/cm) responses approximated isometric responses with minimal shortening (20%). We conclude that elastic loading significantly alters the mechanical properties of airway smooth muscle in vitro, effects that are likely relevant to the loads against which the smooth muscle must contract in vivo.     

An in vitro study of cryopreserved and fresh human arteries: a comparison with ePTFE prostheses and human arteries studied non-invasively in vivo

The surgical options in arterial reconstruction are: the use of autologous arteries; autologous veins; or expanded polytetrafluoroethylene (ePTFE) grafts. However, the development of intimal hyperplasia when using veins or ePTFE grafts has been associated with graft failure. Since autologous arteries are not always available, the use of cryopreserved arteries has to be considered. The aims of this study were: (a) to compare the viscoelastic properties of stored cryopreserved arteries and fresh arteries by in vitro analysis; and (b) to compare the viscoelastic properties of arteries measured non-invasively in normotensive patients, with fresh arteries, cryopreserved arteries, and ePTFE segments. The viscoelastic studies were performed in normotensive patients using stress-strain analysis with non-invasive measurement of pressure and diameter in the common carotid artery, and in vitro measurements of pressure and diameter in arteries and prostheses. The in vitro studies showed that the elastic modulus (E), viscous modulus (eta), Stiffness Index (SI), Peterson modulus (Ep), and the pulse wave velocity (PWV) values for human cryopreserved carotid arteries were similar to the values obtained non-invasively in normotensive subjects (P>0.05) and to human fresh vessels (P>0.05). In vitro, the SI, Ep, PWV, and E values of ePTFE were significantly higher than the observed values in subjects and with fresh and cryopreserved arteries (P<0.05); on the other hand the ePTFE eta values were the lowest (P<0.05). We concluded that cryopreserved arteries have similar viscoelastic properties to those obtained in vivo in the arteries of normotensive subjects and in vitro in fresh arteries. Consequently, we conclude that the cryopreservation procedure does not modify the mechanical properties of the arterial wall.     

An optofluidic mechanical system for elasticity measurement of thin biological tissues

As dura mater has an anisotropic fibrous structure and exists under wet and dynamic stretching conditions in the brain, its mechanical properties have not yet been properly investigated. Here we developed a fluid-assisted mechanical system integrated with a photonic sensor and a pressure sensor in order to measure the elasticity of the dura mater. Porcine dura mater sample was loaded as a stretched diaphragm into a liquid chamber to mimic the in vivo condition. Increasing the flow rate of saline solution into the chamber swelled and deformed the dura mater. The micron-scale deflection of the dura mater was optically detected by the photonic sensor. Fluid pressure and deflection values were then used to calculate the elastic modulus. The average elastic modulus of the porcine dura mater was 31.14 MPa. We further measured the elasticity of a well-known material to further validate the system. We expect that this optofluidic system developed in this study will be useful to measure the elasticity of a variety of thin biological tissues.     

Canine trachealis muscle shortening and cartilage mechanics.

Canine trachealis muscle will shorten by 70% of resting length when maximally stimulated in vitro. In contrast, trachealis muscle will shorten by only 30-40% when stimulated in vivo. To examine the possibility that an elastic load applied by the tracheal cartilage contributes to the in vivo limitation of shortening, single pairs of sonomicrometry crystals were inserted into the trachealis muscle at the level of the fifth cartilage ring in five dogs. The segment containing the crystals was then excised and mounted on a tension-testing apparatus. Points on the active length-tension curve and the passive length-tension relation of the cartilage only were determined. The preload applied to the muscle before contraction varied from 10 to 40 g (mean 21 +/- 4 g). The afterload applied by the cartilage during trachealis contraction ranged from 13 to 56 g (30 +/- 6 g). The calculated elastic afterloads were substantial and appeared to be sufficient to explain the degree of shortening observed in four of the seven rings; in the remaining three rings, the limitation of shortening was greater than would be expected from the elastic load provided by the cartilage. Additional sources of loading and/or additional mechanisms may contribute to limited in situ shortening. In summary, tracheal cartilage applies a preload and an elastic afterload to the trachealis that are substantial and contribute to the limitation of trachealis muscle shortening in vivo.     

Stress wave velocities in bovine patellar tendon.

The velocity of longitudinal stress waves in an elastic body is given by the square root of the ratio of its elastic modulus to its density. In tendinous and ligamentous tissue, the elastic modulus increases with strain and with strain rate. Therefore, it was postulated that stress wave velocity would also increase with increasing strain and strain rate. The purpose of this study was to determine the velocity of stress waves in tendinous tissue as a function of strain and to compare these values to those predicted using the elastic modulus derived from quasi-static testing. Five bovine patellar tendons were harvested and potted as bone-tendon-bone specimens. Quasi-static mechanical properties were determined in tension at a deformation rate of 100 mm/s. Impact loading was employed to determine wave velocity at various strain levels, achieved by preloading the tendon. Following impact, there was a measurable delay in force transmission across the specimen and this delay decreased with increasing tendon strain. The wave velocities at tendon strains of 0.0075, 0.015, and 0.0225 were determined to be 260 +/- 52 m/s, 360 +/- 71 m/s, and 461 +/- 94 m/s, respectively. These velocities were significantly (p < 0.01) faster than those predicted using elastic moduli derived from the quasi-static tests by 52, 45, and 41 percent, respectively. This study has documented that stress wave velocity in patellar tendon increases with increasing strain and is underestimated with a modulus estimated from quasi-static testing.     

In vivo loads on airway smooth muscle: the role of noncontractile airway structures.

The degree of airway smooth muscle contraction and shortening that occurs in vivo is modified by many factors, including those that influence the degree of muscle activation, the resting muscle length, and the loads against which the muscle contracts. Canine trachealis muscle will shorten up to 70% of starting length from optimal length in vitro but will only shorten by around 30% in vivo. This limitation of shortening may be a result of the muscle shortening against an elastic load such as could be applied by tracheal cartilage. Limitation of airway smooth muscle shortening in smaller airways may be the result of contraction against an elastic load, such as could be applied by lung parenchymal recoil. Measurement of the elastic loads applied by the tracheal cartilage to the trachealis muscle and by lung parenchymal recoil to smooth muscle of smaller airways were performed in canine preparations. In both experiments the calculated elastic loads applied by the cartilage and the parenchymal recoil explained in part the limitation of maximal active shortening and airway narrowing observed. We conclude that the elastic loads provided by surrounding structures are important in determining the degree of airway smooth muscle shortening and the resultant airway narrowing.     

Structure and surface energy of the surfactant layer on the alveolar surface: inaccuracies and their correction

The article of Kashchiev and Exerowa (Eur Biophys J 30:34-41) is shown to incorporate a number of inaccuracies that fit the categories "historical", "anatomical", and "biophysical". These inaccuracies are corrected by reference to published research reports from 1978 to 1998. The monolayer-bilayer model proposed by Kashchiev and Exerowa may be thermodynamically correct in vitro, but has not been related to the structure of the alveolar surface in vivo, which is that of a foam ("the alveolar surface network").     

Interactions of 17beta-estradiol and L-norepinephrine on the rat uterus.

Norepinephrine increased the in vitro uptake of 3H-estradiol by the uterus of spayed rats. This effect was observed at 15 and 30 min but not at 90 min. Norepinephrine also increased the binding of 3H-estradiol by the nuclear (p less than 0.02) and the cytosol fractions (p less than 0.01) when incubated with uterine homogenates, suggesting that norepinephrine does not require the presence of the intact tissue to exert its effects. The in vivo uptake of 3H-estradiol and the determination of the number of binding sites were performed in the uterus of rats treated with estradiol and estradiol plus norepinephrine. Norepinephrine alone increased the uptake of 3H-estradiol and the number of binding sites. The highest increment in both parameters was observed in the uterus of rats treated with estradiol plus norepinephrine. The estradiol Ka of the rat uterus cytosol treated with estradiol alone or plus norepinephrine was higher than that observed in the group without estradiol, suggesting the presence of different proteins that bind estradiol. These results indicate that norepinephrine increases the entrance of estradiol into the rat uterus both in vitro and in vivo.     

A simple model describing the elastic properties of human umbilical arterial smooth muscle

Elastic properties of cylindrical segments of 71 normal double and 4 single human umbilical arteries were studied. This specimen is rich in vascular smooth muscle in comparison with other great arteries. Outer diameter versus intraluminal pressure characteristic curves were taken with decreasing pressures in vitro in different contraction states. Tangential force and circumferential incremental elastic modulus were computed. A sum of 222 curves was analysed. The investigations showed that if we take tangential force and outer radius values measured at the same pressure levels but in different contraction states, then a similar proportional change in tangential force will induce a similar proportional passive change in the outer radius, to some extent independently of the degree of active tangential shortening of the segment. For example to induce a 10% passive decrease of the outer radius from values measured at 100 mm Hg intraluminal pressure, tangential force had to be decreased by 76.7 +/- 9.9% in single relaxed arteries, and by 79.6 +/- 0.8% in normal double relaxed segments. These values corresponded to intraluminal pressure levels of 26.4 +/- 4.9 mm Hg and 23.1 +/- 0.9 mm Hg, respectively. In 30% active spontaneous shortening to reach the same 10% passive decrease in outer radius from the value measured at 100 mm Hg, tangential force had to be decreased by 75.2 +/- 1.9% which corresponded to 29.6 +/- 20 mm Hg. The same values in 5-HT induced contraction, 30% active shortening were 76.7 +/- 2.0% and 28.4 +/- 2.6 mm Hg, respectively. In addition to the similarity of relative changes in tangential force, the pressure levels were to some extent also similar. These data suggest that elastic elements in the human umbilical arterial smooth muscle may be organized in such a way as to ensure similar prestretch of similar elastic elements at similar pressures independently of the degree of active shortening of the circumference.     

Elasticity–density and viscoelasticity–density relationships at the tibia mid-diaphysis assessed from resonant ultrasound spectroscopy measurements

Cortical bone tissue is an anisotropic material characterized by typically five independent elastic coefficients (for transverse isotropy) governing shear and longitudinal deformations in the different anatomical directions. It is well established that the Young’s modulus in the direction of the bone axis of long bones has a strong relationship with mass density. It is not clear, however, whether relationships of similar strength exist for the other elastic coefficients, for they have seldom been investigated, and the results available in the literature are contradictory. The objectives of the present work were to document the anisotropic elastic properties of cortical bone at the tibia mid-diaphysis and to elucidate their relationships with mass density. Resonant ultrasound spectroscopy (RUS) was used to measure the transverse isotropic stiffness tensor of 55 specimens from 19 donors. Except for Poisson’s ratios and the non-diagonal stiffness coefficient, strong linear correlations between the different elastic coefficients \((0.7 < {r^{2}} < 0.99)\) and between these coefficients and density \((0.79 < {r^{2}} < 0.89)\) were found. Comparison with previously published data from femur specimens suggested that the strong correlations evidenced in this study may not only be valid for the mid-tibia. RUS also measures the viscous part of the stiffness tensor. An anisotropy ratio close to two was found for damping coefficients. Damping increased as the mass density decreased. The data suggest that a relatively accurate estimation of all the mid-tibia elastic coefficients can be derived from mass density. This is of particular interest (1) to design organ-scale bone models in which elastic coefficients are mapped according to Hounsfield values from computed tomography scans as a surrogate for mass density and (2) to model ultrasound propagation at the mid-tibia, which is an important site for the in vivo assessment of bone status with axial transmission techniques.     

A mechanical model to compute elastic modulus of tissues for harmonic motion imaging

Numerous experimental and computational methods have been developed to estimate tissue elasticity. The existing testing techniques are generally classified into in vitro, invasive in vivo and non-invasive in vivo. For each experimental method, a computational scheme is accordingly proposed to calculate mechanical properties of soft biological tissues. Harmonic motion imaging (HMI) is a new technique that performs radio frequency (RF) signal tracking to estimate the localized oscillatory motion resulting from a radiation force produced by focused ultrasound. A mechanical model and computational scheme based on the superposition principle are developed in this paper to estimate the Young's modulus of a tissue mimicking phantom and bovine liver in vitro tissue from the harmonic displacement measured by HMI. The simulation results are verified by two groups of measurement data, and good agreement is shown in each comparison. Furthermore, an inverse function is observed to correlate the elastic modulus of uniform phantoms with amplitude of displacement measured in HMI. The computational scheme is also implemented to estimate 3D elastic modulus of bovine liver in vitro.     

Morphology of elastic poly(L-lactide-co-epsilon-caprolactone) copolymers and in vitro and in vivo degradation behavior of their scaffolds

Very elastic PLCL [poly(L-lactide-co-epsilon-caprolactone), 50:50] copolymers were synthesized and extruded into porous tubular scaffolds (pore size 150 +/- 50 microm, porosity 90%) for the application to tissue engineering. The copolymers were basically random and amorphous. However, two T(g)'s (glass transition temperatures) were observed in dynamic mechanical thermal analysis and also in differential scanning calorimetry thermograms. Furthermore, microdomains (about 17 nm in size) were indicated on the small-angle X-ray scattering profile and finally confirmed by transmission electron microscopy. Therefore, the PLCL copolymer was probably composed of a soft matrix of mainly epsilon-caprolactone moieties and hard domains containing more L-lactide units to exhibit a rubberlike elasticity in virtue of the physically cross-linked structure. The smooth muscle cells seeded scaffolds were implanted into nude mice subcutaneously for up to 15 weeks to monitor the in vivo degradation. In addition, they were degraded in vitro in phosphate buffer solution (pH 7.4) for up to 1 year to compare the results each other. All the scaffolds degraded slowly in vivo and in vitro even in the form of a highly porous thin membrane. However, the degradation rate was somewhat faster for in vivo than for in vitro. This should be explained by enzymes that might have played a certain role in the degradation in the body. In addition, the epsilon-caprolactone moieties degraded faster than the L-lactide units did in these PLCL scaffolds, although their hydrophilicities are in the opposite order. This behavior appeared more prominently in the in vivo case. This should result from that the amorphous regions composed of mainly epsilon-caprolactone units might have been first attacked by water because water can penetrate into the amorphous regions easier than the hard domains containing more L-lactides.     

Optical measurements of vocal fold tensile properties: implications for phonatory mechanics

In voice research, in vitro tensile stretch experiments of vocal fold tissues are commonly employed to determine the tissue biomechanical properties. In the standard stretch-release protocol, tissue deformation is computed from displacements applied to sutures inserted through the thyroid and arytenoid cartilages, with the cartilages assumed to be rigid. Here, a non-contact optical method was employed to determine the actual tissue deformation of vocal fold lamina propria specimens from three excised human larynges in uniaxial tensile tests. Specimen deformation was found to consist not only of deformation of the tissue itself, but also deformation of the cartilages, as well as suture alignment and tightening. Stress-stretch curves of a representative load cycle were characterized by an incompressible Ogden model. The initial longitudinal elastic modulus was found to be considerably higher if determined based on optical displacement measurements than typical values reported in the literature. The present findings could change the understanding of the mechanics underlying vocal fold vibration. Given the high longitudinal elastic modulus the lamina propria appeared to demonstrate a substantial level of anisotropy. Consequently, transverse shear could play a significant role in vocal fold vibration, and fundamental frequencies of phonation should be predicted by beam theories accounting for such effects.     

The effect of agarose and dexamethasone on the nature and production of extracellular matrix components by elastic cartilage chondrocytes

Chondrocytes isolated enzymatically from rabbit ear cartilage, were cultivated in vitro in the presence of 2% agarose or 0.1 mumol/l dexamethasone. Freshly-isolated chondrocytes suspended in either Eagle's medium or 2% agarose were auto-transplanted intramuscularly. Samples were then examined by light microscopy and transmission electron microscopy. The cells cultivated in vitro rapidly formed confluent multiple overlapping layers filled with a loose matrix consisting of single collagen fibres, proteoglycans and scarce elastic fibres. The number and maturity of the elastic fibres increased substantially after dexamethasone was added. The chondrocytes in intramuscular transplants produced a larger amount of intercellular matrix with many elastic fibres than those cultured in vitro. Addition of agarose to in vitro and in vivo systems selectively suppressed the elastin production but did not diminish the production of elastic fibre microfibrils and other matrix components. This made cultures and transplants of elastic chondrocytes resemble rather hyaline cartilage than the original tissue. It seems that the lack of elastin in the matrix does not result simply from inhibition of elastin secretion or increased elastolysis. It may be related to a reversible change of genetic expression of elastic cartilage chondrocytes under the influence of agarose.     

Mechanical properties of human patellar tendon at the hierarchical levels of tendon and fibril

Tendons are strong hierarchical structures, but how tensile forces are transmitted between different levels remains incompletely understood. Collagen fibrils are thought to be primary determinants of whole tendon properties, and therefore we hypothesized that the whole human patellar tendon and its distinct collagen fibrils would display similar mechanical properties. Human patellar tendons (n = 5) were mechanically tested in vivo by ultrasonography. Biopsies were obtained from each tendon, and individual collagen fibrils were dissected and tested mechanically by atomic force microscopy. The Young's modulus was 2.0 ± 0.5 GPa, and the toe region reached 3.3 ± 1.9% strain in whole patellar tendons. Based on dry cross-sectional area, the Young's modulus of isolated collagen fibrils was 2.8 ± 0.3 GPa, and the toe region reached 0.86 ± 0.08% strain. The measured fibril modulus was insufficient to account for the modulus of the tendon in vivo when fibril content in the tendon was accounted for. Thus, our original hypothesis was not supported, although the in vitro fibril modulus corresponded well with reported in vitro tendon values. This correspondence together with the fibril modulus not being greater than that of tendon supports that fibrillar rather than interfibrillar properties govern the subfailure tendon response, making the fibrillar level a meaningful target of intervention. The lower modulus found in vitro suggests a possible adverse effect of removing the tissue from its natural environment. In addition to the primary work comparing the two hierarchical levels, we also verified the existence of viscoelastic behavior in isolated human collagen fibrils.     

Measurement of the velocity of the pulse wave

Pulse wave transmission rate has been measured "in vivo" in 6 male anaesthetized Wistar rats, by means of cannulae, inserted both in carotid and femoral arteries. The measure has been done by calculating the time delay of several corresponding levels in the two pressure pulses: at the foot, during the ascending phase, at the peak. Three different intervention have been carried out: Ach, NA, blood transfusion. The transmission rate outcame higher at the pulse foot than at the peak, in control animals and in Ach and blood transfusion interventions. The NA intervention inverted this behaviour, where the increase in transmission rate did not correspond to the rate of pressure growing. The possible influences of a vegetative component on arterial viscoelastic behaviour are discussed.     

A fractional derivative model to describe arterial viscoelasticity

Arterial viscoelasticity can be described with a complex modulus (E*) in the frequency domain. In arteries, E* presents a power-law response with a plateau for higher frequencies. Constitutive models based on a combination of purely elastic and viscous elements can be represented with integer order differential equations but show several limitations. Recently, fractional derivative models with fewer parameters have proven to be efficient in describing rheological tissues. A new element, called "spring-pot", that interpolates between springs and dashpots is incorporated. Starting with a Voigt model, we proposed two fractional alternative models with one and two spring-pots. The three models were tested in an anesthetized sheep in a control state and during smooth muscle activation. A least squares method was used to fit E*. Local activation induced a vascular constriction with no pressure changes. The E* results confirmed the steep increase from static to dynamic values and a plateau in the range 2-30 Hz, coherent with fractional model predictions. Activation increased E*, affecting its real and imaginary parts separately. Only the model with two spring-pots correctly followed this behavior with the best performance in terms of least squares errors. In a context where activation separately modifies E*, this alternative model should be considered in describing arterial viscoelasticity in vivo.     

Further characterization of the glucocorticoid-induced antiphospholipase protein "renocortin"

The steroid-induced anti-phospholipase protein "Renocortin" has been further characterized by column chromatography and a monoclonal antibody. In medium devoid of foetal calf serum, renomedullary insterstitial cells in culture exposed to the anti-inflammatory steroid dexamethasone released 3 peptides of apparent MW: 15k, 30k, and 45k possessing similar biological properties to "renocortin", "lipomodulin" and "macrocortin". A monoclonal antibody directed against macrocortin also bound the 45k peptide released from the renomedullary cells. The 15k species was, like macrocortin, inactive in an "in vitro" enzymatic assay but recovered its full inhibitory activity after dephosphorylation by alkaline phosphatase treatment. We conclude that "macrocortin", "lipomodulin" and "renocortin" are similar if not identical proteins. We propose a scheme to account for "in vivo" secretion and regulation of these proteins.