Shape and tension distribution of the active canine diaphragm

Both diaphragm shape and tension contribute to transdiaphragmatic pressure, but of the three variables, tension is most difficult to measure. We measured transdiaphragmatic pressure and the global shape of the in vivo canine diaphragm and used principles of mechanics to compute the tension distribution. Our hypotheses were that 1) tension in the active diaphragm is nonuniform with greater tension in the central tendon than in the muscular regions; 2) maximum tension is essentially oriented in the muscle fiber direction, whereas minimum tension is orthogonal to the fiber direction; and 3) during submaximal activation change in the in vivo global shape is small. Metallic markers, each 2 mm in length, were implanted surgically on the peritoneal surface of the diaphragm at 1.5- to 2.0-cm intervals along the muscle bundles at the midline, ventral, middle, and dorsal regions of the left costal diaphragm and along a muscle bundle of the crural diaphragm. Postsurgery, a biplane videofluoroscopic system was used to determine the in vivo three-dimensional coordinates of the markers at end expiration and end inspiration during quiet breathing as well as at end-inspiratory efforts against an occluded airway at lung volumes of functional residual capacity and at one-third maximum inspiratory capacity increments in volume to total lung capacity. A surface was fit to the marker locations using a two-dimensional spline algorithm. Diaphragm surface was modeled as a pressurized membrane, and tension distribution in the active diaphragm was computed using the ANSYS finite element program. We showed that the peak of the diaphragm dome was closer to the ventral surface than to the dorsal surface and that there was a depression or valley in the crural region. In the supine position, during inspiratory efforts, the caudal displacement of the dorsal region of the diaphragm was greater than that of the dome, and the valley along the crural diaphragm was accentuated. In contrast, at lower lung volumes in the prone posture, the caudal displacement of the dome was greater than that of the crural region. At end of inspiration, transdiaphragmatic pressure was approximately 6.5 cmH2O, and tensions were nonuniform in the diaphragm. Maximum principal stress sigma(1) of central tendon was found to be greater than sigma(1) of the costal region, and that was greater than sigma(1) of the crural region, with values of 14-34, 14-29, and 4-14 g/cm, respectively. The corresponding data of the minimum principal stress sigma(2) were 9-18, 3-9, and 0-1.5 g/cm, respectively. Maximum principal tension was approximately parallel to the muscle fibers, whereas minimum tension was essentially orthogonal to the longitudinal direction of the muscle fibers. In the muscular region, sigma(1) was approximately 3-fold sigma(2), whereas in the central tendon, sigma(1) was only approximately 1.5-fold sigma(2.).     

Passive mechanics of muscle tendinous junction of canine diaphragm.

The diaphragmatic muscle tendon is a biaxially loaded junction in vivo. Stress-strain relations along and transverse to the fiber directions are important in understanding its mechanical properties. We hypothesized that 1) the central tendon possesses greater passive stiffness than adjacent muscle, 2) the diaphragm muscle is anisotropic, whereas the central tendon near the junction is essentially isotropic, and 3) a gradient in passive stiffness exists as one approaches the muscle-tendinous junction (MTJ). To investigate these hypotheses, we conducted uniaxial and biaxial mechanical loading on samples of the MTJ excised from the midcostal region of dog diaphragm. We measured passive length-tension relationships of the muscle, tendon, and MTJ in the direction along the muscle fibers as well as transverse to the fibers. The MTJ was slack in the unloaded state, resulting in a J-shaped passive tension-strain curve. Generally, muscle strain was greater than that of MTJ, which was greater than tendon strain. In the muscular region, stiffness in the direction transverse to the fibers is much greater than that along the fibers. The central tendon is essentially inextensible in the direction transverse to the fibers as well as along the fibers. Our data demonstrate the existence of more pronounced anisotropy in the muscle than in the tendon near the junction. Furthermore, a gradient in muscle stiffness exists as one approaches the MTJ, consistent with the hypothesis of continuous passive stiffness across the MTJ.     

Effects of transverse fiber stiffness and central tendon on displacement and shape of a simple diaphragm model

Boriek, Aladin M., and Joseph R. Rodarte. Effects oftransverse fiber stiffness and central tendon on displacement and shapeof a simple diaphragm model. J. Appl. Physiol. 82(5): 1626-1636, 1997.Our previous experimental results (A. M. Boriek, S. Lui, and J. R. Rodarte. J. Appl. Physiol. 75:527-533, 1993 and A. M. Boriek, T. A. Wilson, and J. R. Rodarte.J. Appl. Physiol. 76: 223-229, 1994) showed that1) costal diaphragm shape is similar at functional residualcapacity and end inspiration regardless of whether the diaphragm muscleshortens actively (increased tension) or passively (decreased tension);2) diaphragmatic muscle length changes minimally in thedirection transverse to the muscle fibers, suggesting the diaphragm maybe inextensible in that direction; and 3) the central tendon isnot stretched by physiological stresses. A two-dimensional orthotropicmaterial has two different stiffnesses in orthogonal directions. In theplane tangent to the muscle surface, these directions are along thefibers and transverse to the fibers. We wondered whether orthotropicmaterial properties in the muscular region of the diaphragm andinextensibility of the central tendon might contribute to the constancyof diaphragm shape. Therefore, in the present study, we examined theeffects of stiffness transverse to muscle fibers and inextensibility ofthe central tendon on diaphragmatic displacement and shape. Finiteelement hemispherical models of the diaphragm were developed by usingpressurized isotropic and orthotropic membranes with a wide range ofstiffness ratios. We also tested heterogeneous models, in which themuscle sheet was an orthotropic material, having transverse fiberstiffness greater than that along the fibers, with the central tendonbeing an inextensible isotropic cap. These models revealed thatincreased transverse stiffness limits the shape change of thediaphragm. Furthermore, an inextensible cap simulating the centraltendon dramatically limits the change in shape as well as the membrane displacement in response to pressure. These findings provide a plausible mechanism by which the diaphragm maintains similar shapes despite different physiological loads. This study suggests that changesof diaphragm shape are restricted because the central tendon isessentially inextensible and stiffness in the direction transverse tothe muscle fibers is greater than stiffness along the fibers.

     

Tensile properties and fiber alignment of human supraspinatus tendon in the transverse direction demonstrate inhomogeneity,nonlinearity, and regional isotropy

A recent study (Lake et al., 2009); reported the properties of human supraspinatus tendon (SST) tested along the predominant fiber direction. The SST was found to have a relatively disperse distribution of collagen fibers, which may represent an adaptation to multiaxial loads imposed by the complex loading environment of the rotator cuff. However, the multiaxial mechanical properties of human SST remain unknown. The objective of this study, therefore, was to evaluate the mechanical properties, fiber alignment, change in alignment with applied load, and structure–function relationships of SST in transverse testing. Samples from six SST locations were tested in uniaxial tension with samples oriented transverse to the tendon long-axis. Polarized light imaging was used to quantify collagen fiber alignment and change in alignment under applied load. The mechanical properties of samples taken near the tendon–bone insertion were much greater on the bursal surface compared to the joint surface (e.g., bursal moduli 15–30 times greater than joint; p<0.001). In fact, the transverse moduli values of the bursal samples were very similar to values obtained from samples tested along the tendon long-axis (Lake et al., 2009). This key and unexpected finding suggests planar mechanical isotropy for bursal surface samples near the insertion, which may be due to complex in vivo loading. Organizationally, fiber distributions became less aligned along the tendon long-axis in the toe-region of the stress–strain response. Alignment changes occurred to a slightly lesser degree in the linear-region, suggesting that movement of collagen fibers may play a role in mechanical nonlinearity. Transverse mechanical properties were significantly correlated with fiber alignment (e.g., for linear-region modulus r_s=0.74, p<0.0001), demonstrating strong structure–function relationships. These results greatly enhance current understanding of the properties of human SST and provide clinicians and scientists with vital information in attempting to treat or replace this complex tissue.     

Inferences on force transmission from muscle fiber architecture of the canine diaphragm

Functional properties of the diaphragm are mediated by muscle structure. Modeling of force transmission necessitates a precise knowledge of muscle fiber architecture. Because the diaphragm experiences loads both along and transverse to the long axes of its muscle fibers in vivo, the mechanism of force transmission may be more complex than in other skeletal muscles that are loaded uniaxially along the muscle fibers. Using a combination of fiber microdissections and histological and morphological methods, we determined regional muscle fiber architecture and measured the shape of the cell membrane of single fibers isolated from diaphragm muscles from 11 mongrel dogs. We found that muscle fibers were either spanning fibers (SPF), running uninterrupted between central tendon (CT) and chest wall (CW), or were non-spanning fibers (NSF) that ended within the muscle fascicle. NSF accounted for the majority of fibers in the midcostal, dorsal costal, and lateral crural regions but were only 25-41% of fibers in the sternal region. In the midcostal and dorsal costal regions, only approximately 1% of the NSF terminated within the fascicle at both ends; the lateral crural region contained no such fibers. We measured fiber length, tapered length, fiber diameters along fiber length, and the taper angle for 271 fibers. The lateral crural region had the longest mean length of SPF, which is equivalent to the mean muscle length, followed by the costal and sternal regions. For the midcostal and crural regions, the percentage of tapered length of NSF was 45.9 +/- 5.3 and 40.6 +/- 7.5, respectively. The taper angle was approximately 0.15 degrees for both, and, therefore, the shear component of force was approximately 380 times greater than the tensile component. When the diaphragm is submaximally activated, as during normal breathing and maximal inspiratory efforts, muscle forces could be transmitted to the cell membrane and to the extracellular intramuscular connective tissue by shear linkage, presumably via structural transmembrane proteins.     

Respiratory changes in diaphragmatic intramuscular pressure

We attempted to measure diaphragmatic tension by measuring changes in diaphragmatic intramuscular pressure (Pim) in the costal and crural parts of the diaphragm in 10 supine anesthetized dogs with Gaeltec 12 CT minitransducers. During phrenic nerve stimulation or direct stimulation of the costal and crural parts of the diaphragm in an animal with the chest and abdomen open, Pim invariably increased and a linear relationship between Pim and the force exerted on the central tendon was found (r greater than or equal to 0.93). During quiet inspiration Pim in general decreased in the costal part (-3.9 +/- 3.3 cmH2O), whereas it either increased or slightly decreased in the crural part (+3.3 +/- 9.4 cmH2O, P less than 0.05). Similar differences were obtained during loaded and occluded inspiration. After bilateral phrenicotomy Pim invariably decreased during inspiration in both parts (costal -4.3 +/- 6.4 cmH2O, crural -3.1 +/- 0.6 cmH2O). Contrary to the expected changes in tension in the muscle, but in conformity with the pressure applied to the muscle, Pim invariably increased during passive inflation from functional residual capacity to total lung capacity (costal +30 +/- 23 cmH2O, crural +18 +/- 18 cmH2O). Similarly, during passive deflation from functional residual capacity to residual volume, Pim invariably decreased (costal -12 +/- 19 cmH2O, crural -12 +/- 14 cmH2O). In two experiments similar observations were made with saline-filled catheters. We conclude that although Pim increases during contraction as in other muscles, Pim during respiratory maneuvers is primarily determined by the pleural and abdominal pressures applied to the muscle rather than by the tension developed by it.     

Fiber architecture of canine abdominal muscles.

During respiration, abdominal muscles experience loads, not only in the muscle-fiber direction but also transverse to the fibers. We wondered whether the abdominal muscles exhibit a fiber architecture that is similar to the diaphragm muscle, and, therefore, we chose two adjacent muscles: the internal oblique (IO), with about the same muscle length as the diaphragm, and the transverse abdominis (TA), which is twice as long as the diaphragm. First, we used acetylcholinesterase staining to examine the distribution of neuromuscular junctions on both surfaces of the TA and IO muscles in six dogs. A maximum of four irregular bands of neuromuscular junctions crossed the IO, and as many as six bands crossed the TA, which is consistent with a discontinuous fiber architecture. In six additional dogs, we examined fiber architecture of these muscles by microdissecting 103 fascicles from the IO and 139 from the TA. Each fascicle contained between 20 and 30 muscle fibers. The mean length of nonspanning fibers (NSF) ranged from 2.8 +/- 0.3 cm in the IO to 4.3 +/- 0.5 cm in the TA, and the mean length of spanning fibers ranged from 4.3 +/- 0.5 cm in the IO to 7.6 +/- 1.4 cm in the TA. NSF accounted for 89.6 +/- 1.5% of all fibers dissected from the IO and 99.1 +/- 0.2% of all fibers dissected from the TA. The percentage of NSF with both ends tapered was 6.2 +/- 1.0 and 41.0 +/- 2.3% for IO and TA, respectively. These data show that fiber architecture in either IO or TA is discontinuous, with much more short-tapered fibers in the TA than in the IO. When abdominal muscles are submaximally activated, as during both normal expiration and maximal expiratory efforts, muscle force could be transmitted to the cell membrane and to the extracellular intramuscular connective tissue by shear linkage, presumably via structural transmembrane proteins.     

Mechanical implications of in vivo human diaphragm shape.

Using magnetic resonance imaging, we measured the three-dimensional form of the diaphragm in vivo in four supine relaxed subjects at functional residual capacity and calculated its total surface area, the right and left surface areas in the zone of apposition, and the principal radii of curvature as a function of height. The area of apposition comprised 45 +/- 1.5% (SE) of the total surface area of the diaphragm. Available data on the area of the central tendon indicate that a considerable part of the muscular part of the diaphragm is lung apposed. The curvature was linearly related to height over 7 cm of the posterior half of each hemidiaphragm. From the linear portion of this graph and assuming a vertical gradient of transdiaphragmatic pressure of 0.75 cmH2O/cm, we applied the Laplace law and calculated tensions of 54 and 32 g/cm for right and left sides, respectively. We conclude that the shape of at least part of the posterior half of the relaxed human diaphragm in the supine position at functional residual capacity can be explained by the Laplace law, suggesting that both the lung and abdominal contents behave sufficiently as fluids so that they do not impose their shape on the diaphragm. Because diaphragm muscle is partly lung apposed, it is unlikely that the diaphragm functions simply as a piston.     

Efficiency of the normal human diaphragm with hyperinflation.

We evaluated an index of diaphragm efficiency (Eff(di)), diaphragm power output (Wdi) relative to electrical activation, in five healthy adults during tidal breathing at usual end-expiratory lung volume (EELV) and diaphragm length (L(di ee)) and at shorter L(di ee) during hyperinflation with expiratory positive airway pressure (EPAP). Measurements were repeated with an inspiratory threshold (7.5 cmH(2)O) plus resistive (6.5 cmH(2)O.l(-1).s) load. Wdi was the product of mean inspiratory transdiaphragmatic pressure (DeltaPdi(mean)), diaphragm volume displacement measured fluoroscopically, and 1/inspiratory duration (Ti(-1)). Diaphragm activation, measured with esophageal electrodes, was quantified by computing root-mean-square values (RMS(di)). With EPAP, 1) EELV increased [mean r(2) = 0.91 (SD 0.01)]; 2) in four subjects, L(di ee) decreased [mean r(2) = 0.85 (SD 0.07)] and mean Eff(di) decreased 34% per 10% decrease in L(di ee) (P < 0.001); and 3) in one subject, gastric pressure at EELV increased two- to threefold, L(di ee) was unchanged or increased, and Eff(di) increased at two of four levels of EPAP (P < or = 0.006, ANOVA). Inspiratory loading increased Wdi (P = 0.003) and RMS(di) (P = 0.004) with no change in Eff(di) (P = 0.63) or its relationship with L(di ee). Eff(di) was more accurate in defining changes in L(di ee) [(true positives + true negatives)/total = 0.78 (SD 0.13)] than DeltaPdi(mean).RMS(di)(-1), RMS(di), or DeltaPdi(mean).Ti (all <0.7, P < or = 0.05, without load). Thus Eff(di) was principally a function of L(di ee) independent of inspiratory loading, behavior consistent with muscle force-length-velocity properties. We conclude that Eff(di), measured during tidal breathing and in the absence of expiratory muscle activity at EELV, is a valid and accurate measure of diaphragm contractile function.     

Passive mechanics of canine internal abdominal muscles.

The internal abdominal muscles are biaxially loaded in vivo, and therefore length-tension relations along and transverse to the directions of the muscle fibers are important in understanding their mechanical properties. We hypothesized that 1) internal oblique and transversus abdominis form an internal abdominal composite muscle with altered compliance than that of either muscle individually, and 2) anisotropy, different compliances in orthogonal directions, of internal abdominal composite muscle is less pronounced than that of its individual muscles. To test these hypotheses, in vitro mechanical testing was performed on 5 x 5 cm squares of transversus abdominis, internal oblique, and the two muscles together as a composite. These tissues were harvested from the left lateral side of abdominal muscles of eleven mongrel dogs (15-23 kg) and placed in a bath of oxygenated Krebs solution. Each tissue strip was attached to a biaxial mechanical testing device. Each muscle was passively lengthened and shortened along muscle fibers, transverse to fibers, or simultaneously along and transverse to muscle fibers. Both transversus abdominis and internal oblique muscles demonstrated less extensibility in the direction transverse to muscle fibers than along fibers. Biaxial loading caused a stiffening effect that was greater in the direction along the fibers than transverse to the fibers. Furthermore, the abdominal muscle composite was less compliant than either muscle alone in the direction of the muscle fibers. Taken together, our data suggested that the internal abdominal composite tissue has complex mechanical properties that are dependent on the mechanical properties of internal oblique and transversus abdominis muscles.     

Effect of fiber orientation and strain rate on the nonlinear uniaxial tensile material properties of tendon

Tendons are exposed to complex loading scenarios that can only be quantified by mathematical models, requiring a full knowledge of tendon mechanical properties. This study measured the anisotropic, nonlinear, elastic material properties of tendon. Previous studies have primarily used constant strain-rate tensile tests to determine elastic modulus in the fiber direction. Data for Poisson's ratio aligned with the fiber direction and all material properties transverse to the fiber direction are sparse. Additionally, it is not known whether quasi-static constant strain-rate tests represent equilibrium elastic tissue behavior. Incremental stress-relaxation and constant strain-rate tensile tests were performed on sheep flexor tendon samples aligned with the tendon fiber direction or transverse to the fiber direction to determine the anisotropic properties of toe-region modulus (E0), linear-region modulus (E), and Poisson's ratio (v). Among the modulus values calculated, only fiber-aligned linear-region modulus (E1) was found to be strain-rate dependent. The E1 calculated from the constant strain-rate tests were significantly greater than the value calculated from incremental stress-relaxation testing. Fiber-aligned toe-region modulus (E(1)0 = 10.5 +/- 4.7 MPa) and linear-region modulus (E1 = 34.0 +/- 15.5 MPa) were consistently 2 orders of magnitude greater than transverse moduli (E(2)0 = 0.055 +/- 0.044 MPa, E2 = 0.157 +/- 0.154 MPa). Poisson's ratio values were not found to be rate-dependent in either the fiber-aligned (v12 = 2.98 +/- 2.59, n = 24) or transverse (v21 = 0.488 +/- 0.653, n = 22) directions, and average Poisson's ratio values in the fiber-aligned direction were six times greater than in the transverse direction. The lack of strain-rate dependence of transverse properties demonstrates that slow constant strain-rate tests represent elastic properties in the transverse direction. However, the strain-rate dependence demonstrated by the fiber-aligned linear-region modulus suggests that incremental stress-relaxation tests are necessary to determine the equilibrium elastic properties of tendon, and may be more appropriate for determining the properties to be used in elastic mathematical models.     

On the anisotropy of the canine diaphragmatic central tendon

We studied the mechanical and anatomical anisotropy of the canine diaphragmatic central tendon (CT). Dumb-bell-shaped strips with effective dimensions of 10 x 2 mm (length x width) were cut from different regions of the canine diaphragmatic CT in two different orientations relative to the direction of neighboring muscle fibers. Specimens sampled with their long axial dimension oriented parallel to the neighboring muscle fibers were named Group-1 and those sampled with an orientation perpendicular to the neighboring muscle fibers were named Group-2. Results from one-dimensional stress-strain and tensile failure strength tests revealed that the CT is a nonlinear, inelastic, and anisotropic material. Group-1 specimens were found to have a higher stiffness, higher failure strength and higher strain energy density at failure than Group-2 specimens. Polarized microscopy showed that multiple sheets of collagen fiber bundles formed an orthogonal network in the tendon. Collagen fiber bundles along Group-1 direction formed parallel trajectory lines connecting the neighboring costal and crural muscles; bundles along Group-2 direction were observed to orient 90 degrees away. At the central apex region of the CT, collagen bundles of Group-1 formed a fan-like trajectory pattern. This collagen network architecture was compared favorably to the trajectories of an approximated principal stress field in the CT due to simulated contractile forces from its adjacent costal and crural muscles. These combined results suggest a structure-function relationship for the anatomical and mechanical anisotropy in the canine diaphragmatic CT.     

Muscle fiber architecture of the dog diaphragm

Boriek, Aladin M., Charles C. Miller III, and Joseph R. Rodarte. Muscle fiber architecture of the dog diaphragm.J. Appl. Physiol. 84(1): 318-326, 1998.Previous measurements of muscle thickness and length ratio ofcostal diaphragm insertions in the dog (A. M. Boriek and J. R. Rodarte.J. Appl. Physiol. 77: 2065-2070,1994) suggested, but did not prove, discontinuous muscle fiberarchitecture. We examined diaphragmatic muscle fiber architecture usingmorphological and histochemical methods. In 15 mongrel dogs, transversesections along the length of the muscle fibers were analyzedmorphometrically at ×20, by using the BioQuant System IVsoftware. We measured fiber diameters, cross-sectional fiber shapes,and cross-sectional area distributions of fibers. We also determinednumbers of muscle fibers per cross-sectional area and ratio ofconnective tissue to muscle fibers along a course of the muscle fromnear the chest wall (CW) to near the central tendon (CT) for midcostalleft and right hemidiaphragms, as well as ventral, middle, and dorsalregions of the left costal hemidiaphragm. In six other mongrel dogs,the macroscopic distribution of neuromuscular junctions (NMJ) onthoracic and abdominal diaphragm surfaces was determined by stainingthe intact diaphragmatic muscle for acetylcholinesterase activity. Theaverage major diameter of muscle fibers was significantly smaller, andthe number of fibers was significantly larger midspan between CT and CWthan near the insertions. The ratio of connective tissues to musclefibers was largest at CW compared with other regions along the lengthof the muscle. The diaphragm is transversely crossed by multiplescattered NMJ bands with fairly regular intervals offset in adjacentstrips. Muscle fascicles traverse two to five NMJ, consistent withfibers that do not span the entire fascicle from CT to CW. Theseresults suggest that the diaphragm has a discontinuous fiberarchitecture in which contractile forces may be transmitted among themuscle fibers through the connective tissue adjacent to the fibers.

     

A biphasic and transversely isotropic mechanical model for tendon: application to mouse tail fascicles in uniaxial tension

A transversely isotropic biphasic mixture model was applied to tendon in uniaxial tension. Parametric analyses were performed and the sensitivity in predicting material parameters was evaluated. Our results provide quantitative evidence for fluid flow as a mechanism that contributes to tendon viscoelasticity. Transversely isotropic material properties were calculated for mouse tail tendon fascicles. The average transverse modulus (E(1)) was 0.046 MPa, the fiber-aligned Poisson's ratio (v(31)) was 2.73, and the transverse Poisson's ratio [(v(21)) was 0.96; these properties were not strain-dependent. The fiber-aligned modulus (E(s)) was strain-dependent and was 20.7 MPa in the toe region and 86.1 MPa in the linear region. These solid matrix properties were consistent with previously published tendon tissue and fascicle data. The fascicle permeability was strain-dependent and was 5.5 x 10(-18)m(4)/Ns in the toe region and 0.32 x 10(-18)m(4)/Ns in the linear region, similar to previously reported meniscus permeability in tension. The similar permeabilities of both fascicle and tissue-level samples suggest that fluid flow from individual fascicles, not the packing of multiple fascicles together, may be the primary barrier to fluid flow in tendon and thus the primary mechanism for viscoelasticity.     

Ultrastructure of clots during isometric contraction

We explored the retraction or contraction of platelet-fibrin clots under isometric conditions. In the presence of micromolar calcium clots of normal platelet-rich plasma developed tension at an initial rate of 0.1 to 0.2 g/min per cm2 (initial cross-sectional area). Electron microscopy of clots fixed after attaining a force of 1.6 g/cm2 revealed platelets with elongated bodies and pseudopods in close apposition to fibrin strands which were oriented in cablelike fashion in the direction of tension. The development of tension could not be explained simply on the basis of platelet-platelet association and interaction alone. First, factor XIII-dependent cross-linking of fibrin fibers was critical to normal isometric contraction. Second, tension decreased linearly, rather than exponentially, when the platelet count in the platelet-fibrin clot was decreased, suggesting that platelets must be interacting with another component (i.e. fibrin). Thrombasthenic platelets, deficient in fibrinogen receptors, failed to develop tension or to align fibrin strands or pseudopods in the clot. Platelet-fibrin clots treated with vincristine to disassemble microtubules or cytochalasin B to disrupt microfilaments failed to develop tension and relaxed if these agents were added after tension had developed. Relaxation under these conditions, however, was not associated with loss of orientation of fibrin strands. Our findings suggest that platelet-fibrin interaction in clots under isometric conditions leads to orientation of fibrin strands and platelets in the direction of force generation. Tension develops as platelets simultaneously attach to and spread along fibrin strands, and contract. The contraction draws some fibrin into platelet-fibrin clumps and aligns other strands in the long axis of tension. The achievement and maintenance of maximum tension appears to depend on the development of platelet-fibrin attachments and extension of platelet bodies and long pseudopods containing bundles of microfilaments and microtubules along the oriented fibrin fibers.     

Biaxial tensile testing and constitutive modeling of human supraspinatus tendon

The heterogeneous composition and mechanical properties of the supraspinatus tendon offer an opportunity for studying the structure-function relationships of fibrous musculoskeletal connective tissues. Previous uniaxial testing has demonstrated a correlation between the collagen fiber angle distribution and tendon mechanics in response to tensile loading both parallel and transverse to the tendon longitudinal axis. However, the planar mechanics of the supraspinatus tendon may be more appropriately characterized through biaxial tensile testing, which avoids the limitation of nonphysiologic traction-free boundary conditions present during uniaxial testing. Combined with a structural constitutive model, biaxial testing can help identify the specific structural mechanisms underlying the tendon's two-dimensional mechanical behavior. Therefore, the objective of this study was to evaluate the contribution of collagen fiber organization to the planar tensile mechanics of the human supraspinatus tendon by fitting biaxial tensile data with a structural constitutive model that incorporates a sample-specific angular distribution of nonlinear fibers. Regional samples were tested under several biaxial boundary conditions while simultaneously measuring the collagen fiber orientations via polarized light imaging. The histograms of fiber angles were fit with a von Mises probability distribution and input into a hyperelastic constitutive model incorporating the contributions of the uncrimped fibers. Samples with a wide fiber angle distribution produced greater transverse stresses than more highly aligned samples. The structural model fit the longitudinal stresses well (median R(2) ≥ 0.96) and was validated by successfully predicting the stress response to a mechanical protocol not used for parameter estimation. The transverse stresses were fit less well with greater errors observed for less aligned samples. Sensitivity analyses and relatively affine fiber kinematics suggest that these errors are not due to inaccuracies in measuring the collagen fiber organization. More likely, additional strain energy terms representing fiber-fiber interactions are necessary to provide a closer approximation of the transverse stresses. Nevertheless, this approach demonstrated that the longitudinal tensile mechanics of the supraspinatus tendon are primarily dependent on the moduli, crimp, and angular distribution of its collagen fibers. These results add to the existing knowledge of structure-function relationships in fibrous musculoskeletal tissue, which is valuable for understanding the etiology of degenerative disease, developing effective tissue engineering design strategies, and predicting outcomes of tissue repair.     

Redox modulation of diaphragm contractility: Interaction between DHPR and RyR channels

Previous reports indicate that reactive oxygen species (ROS) may modulate contractility in skeletal muscle. Although Ca(2+)-sensitivity of the contractile apparatus appears to be a primary site of regulation, dihydropyridine receptor (DHPR or L-type Ca(2+) channels) and calcium efflux in isolated sarcoplasmic reticulum (SR) vesicles appear to be redox sensitive as well. However, DHPR as a target is poorly understood in intact muscles at body temperature, particularly in the diaphragm, a muscle more dependent on external Ca(2+) than locomotor muscles. Previously, we reported that oxidant challenge via xanthine oxidase (XO) alters the K(+) contractures in diaphragm fiber bundles, suggestive of a role of L-type Ca(2+) channels. Contractility of isolated rat diaphragm fiber bundles revealed a biphasic response to ROS challenge that was dose and time dependent. Potentiation of twitch and low-frequency diaphragm fiber bundle contractility with 0.02 U?ml(-1) XO was reversible or partially preventable with washout, dithiothreitol, and the SOD/catalase mimetic EUK-134. The RyR antagonist ruthenium red inhibited xanthine oxidase-induced potentiation, while the RyR agonist caffeine elevated diaphragm twitch and low-frequency tension in a non-additive manner by 55% when introduced simultaneously with ROS challenge. The DHPR antagonist nitrendipine (15 μM) inhibited elevation in low-frequency diaphragm tension produced by ROS challenge. Caffeine threshold tension curves were shifted to the left with 0.02 U?ml(-1) XO, but this effect was partially reversed with 15 μM nitrendipine. These results are consistent with the hypothesis that DHPR redox state and RyR function are modulated in an interactive manner, affecting contractility in intact diaphragm fiber bundles.     

A DECODER NMR study of backbone orientation in Nephila clavipes dragline silk under varying strain and draw rate

Using DECODER (direction exchange with correlation for orientation distribution evaluation and reconstruction) NMR, we probe the orientations of carbonyl carbons in [1-(13)C]glycine-labeled dragline silk under conditions of varying strain and fiber draw rate. A model-specific reconstruction of the molecular orientation distribution incorporating beta sheets and polyglycine II helices indicates that the structures' alignment along the fiber can be described by a pair of Gaussian distributions with full width at half-maxima of 20 and 68 degrees and approximately 45 and approximately 55% relative contributions to the signal intensity. The alignment along the fiber was found to change appreciably when the drawing tension on the fiber was relaxed in a sample drawn at 4 cm/s while little change was observed in a sample drawn at 2 cm/s. The degree of alignment along the fiber was found to increase with fiber draw rate.     

Liquid drainage through the peritoneal diaphragmatic surface

In 14 spontaneously breathing anesthetized rabbits, we used cyanoacrylate to glue a hollow capsule, at end expiration or at end inspiration, to the peritoneal surface of the tendinous portion of the diaphragm. The capsule was connected to a pressure transducer and a pipette calibrated in microliters. We filled the system with fluid and measured flow into the diaphragmatic surface facing the capsule (Fcap, microliter/cm2), from liquid displacement in the pipette at different hydraulic pressures in the system (Pcap). Pleural liquid pressure was simultaneously measured in the supraphrenic region (Psup). Fcap was positively correlated to transdiaphragmatic pressure gradient (Psup-Pcap) and breathing frequency but was unaffected by protein concentration of capsular fluid. For a breathing frequency of 30 cycles/min and a Psup - Pcap = -2 cmH2O, Fcap was 0.54 microliter.min-1.cm-2 for capsules applied at end expiration and 10-fold greater for capsules applied at end inspiration. Data indicate that the diaphragmatic tendinous portion in rabbits is a draining site for peritoneal fluid and that the conductance of the draining pathways (lymphatic stomata) is related to diaphragmatic tension. In the intact rabbit the average peritoneal fluid drainage through the tendinous portion of the diaphragm (approximately 16 cm2) was estimated at 43 microliters/min.     

Functional arrangement of rat diaphragmatic initial lymphatic network

Fluid and solute flux between the pleural and peritoneal cavities, although never documented under physiological conditions, might play a relevant role in pathological conditions associated with the development of ascitis and pleural effusion and/or in the processes of tumor dissemination. To verify whether a pleuroperitoneal flux might take place through the diaphragmatic lymphatic network, the transdiaphragmatic pressure gradient (Delta P(TD)) was measured in five spontaneously breathing anesthetized rats. Delta P(TD) was -1.93 cmH2O (SD 0.59) and -3.1 cmH2O (SD 0.82) at end expiration and at end inspiration, respectively, indicating the existence of a pressure gradient directed from the abdominal to the pleural cavity. Morphometrical analysis of the diaphragmatic lymphatic network was performed in the excised diaphragm of three additional rats euthanized with an anesthesia overdose. Optical and electron microscopy revealed that lymphatic submesothelial lacunae and lymphatic capillaries among the skeletal muscles fibers show the ultrastructural features of the so-called initial lymphatic vessels, namely, a discontinuous basal lamina and anchoring filaments linking the outer surface of the endothelial cells to connective tissue or to muscle fibers. Primary unidirectional valves in the wall of the initial lymphatics allow entrance of serosal fluid into the lymphatic network preventing fluid backflow, while unidirectional intraluminar valves in the transverse vessels convey lymph centripetally toward central collecting ducts. The complexity and anatomical arrangement of the two valves system suggests that, despite the existence of a favorable Delta P(TD), in the physiological condition no fluid bulk flow takes place between the pleural and peritoneal cavity through the diaphragmatic lymphatic network.