Andreas J. Schriefl

Determination of the layer-specific distributed collagen fibre orientations in human thoracic and abdominal aortas and common iliac arteries

The established method of polarized microscopy in combination with a universal stage is used to determine the layer-specific distributed collagen fibre orientations in 11 human non-atherosclerotic thoracic and abdominal aortas and common iliac arteries (63 ± 15.3 years, mean ± s.d.). A dispersion model is used to quantify over 37 000 recorded fibre angles from tissue samples. The study resulted in distinct fibre families, fibre directions, dispersion and thickness data for each layer and all vessels investigated. Two fibre families were present for the intima, media and adventitia in the aortas, with often a third and sometimes a fourth family in the intima in the respective axial and circumferential directions. In all aortas, the two families were almost symmetrically arranged with respect to the cylinder axis, closer to the axial direction in the adventitia, closer to the circumferential direction in the media and in between in the intima. The same trend was found for the intima and adventitia of the common iliac arteries; however, there was only one preferred fibre alignment present in the media. In all locations and layers, the observed fibre orientations were always in the tangential plane of the walls, with no radial components and very small dispersion through the wall thickness. A wider range of in-plane fibre orientations was present in the intima than in the media and adventitia. The mean total wall thickness for the aortas and the common iliac artery was 1.39 and 1.05 mm, respectively. For the aortas, a slight thickening of the intima and a thinning of the media in increasingly distal regions were observed. A clear intimal thickening was present distal to the branching of the celiac arteries. All data, except for the media of the common iliac arteries, showed two prominent collagen fibre families for all layers so that two-fibre family models seem most appropriate.

Modelling non-symmetric collagen fibre dispersion in arterial walls

New experimental results on collagen fibre dispersion in human arterial layers have shown that the dispersion in the tangential plane is more significant than that out of plane. A rotationally symmetric dispersion model is not able to capture this distinction. For this reason, we introduce a new non-symmetric dispersion model, based on the bivariate von Mises distribution, which is used to construct a new structure tensor. The latter is incorporated in a strain-energy function that accommodates both the mechanical and structural features of the material, extending our rotationally symmetric dispersion model (Gasser et al. 2006 J. R. Soc. Interface 3, 15–35. (doi:10.1098/rsif.2005.0073)). We provide specific ranges for the dispersion parameters and show how previous models can be deduced as special cases. We also provide explicit expressions for the stress and elasticity tensors in the Lagrangian description that are needed for a finite-element implementation. Material and structural parameters were obtained by fitting predictions of the model to experimental data obtained from human abdominal aortic adventitia. In a finite-element example, we analyse the influence of the fibre dispersion on the homogeneous biaxial mechanical response of aortic strips, and in a final example the non-homogeneous stress distribution is obtained for circumferential and axial strips under fixed extension. It has recently become apparent that this more general model is needed for describing the mechanical behaviour of a variety of fibrous tissues.

Biomechanical properties and microstructure of human ventricular myocardium

UNLABELLED In the multidisciplinary field of heart research it is of utmost importance to identify accurate myocardium material properties for the description of phenomena such as mechano-electric feedback or heart wall thickening. A rationally-based material model is required to understand the highly nonlinear mechanics of complex structures such as the passive myocardium under different loading conditions. Unfortunately, to date there are no experimental data of human heart tissues available to estimate material parameters and to develop adequate material models. This study aimed to determine biaxial extension and triaxial shear properties and the underlying microstructure of the passive human ventricular myocardium. Using new state-of-the-art equipment, planar biaxial extension tests were performed to determine the biaxial extension properties of the passive ventricular human myocardium. Shear properties of the myocardium were examined by triaxial simple shear tests performed on small cubic specimens excised from an adjacent region of the biaxial extension specimens. The three-dimensional microstructure was investigated through second-harmonic generation (SHG) microscopy on optically cleared tissues, which emphasized the 3D orientation and dispersion of the myofibers and adjacent collagen fabrics. The results suggest that the passive human LV myocardium under quasi-static and dynamic multiaxial loadings is a nonlinear, anisotropic (orthotropic), viscoelastic and history-dependent soft biological material undergoing large deformations. Material properties of the tissue components along local microstructural axes drive the nonlinear and orthotropic features of the myocardium. SHG microscopy investigation revealed detailed information about the myocardial microstructure due to its high resolution. It enabled the identification of structural parameters such as the fiber and the sheet orientations and corresponding dispersions. With this complete set of material data, a sophisticated material model and associated material parameters can be defined for a better description of the biomechanical response of the ventricular myocardium in humans. Such a model will lead to more accurate computational simulations to better understand the fundamental underlying ventricular mechanics, a step needed in the improvement of medical treatment of heart diseases. STATEMENT OF SIGNIFICANCE Unfortunately, to date there are no experimental data of human heart tissues available for material parameter estimation and the development of adequate material models. In this manuscript novel biaxial tensile and shear test data at different specimen orientations are presented, which allowed to adequately capture the direction-dependent material response. With these complete sets of mechanical data, combined with their underlying microstructural data (also presented herein), sophisticated material models and associated material parameters can be defined for the description of the mechanical behavior of the ventricular myocardium in humans. Such models will lead to accurate computational simulations to better understand the fundamental underlying ventricular mechanics, a step needed in the improvement of medical treatment of heart diseases.

An automated approach for three-dimensional quantification of fibrillar structures in optically cleared soft biological tissues.

We present a novel approach allowing for a simple, fast and automated morphological analysis of three-dimensional image stacks (z-stacks) featuring fibrillar structures from optically cleared soft biological tissues. Five non-atherosclerotic tissue samples from human abdominal aortas were used to outline the multi-purpose methodology, applicable to various tissue types. It yields a three-dimensional orientational distribution of relative amplitudes, representing the original collagen fibre morphology, identifies regions of isotropy where no preferred fibre orientations are observed and determines structural parameters throughout anisotropic regions for the analysis and numerical modelling of biomechanical quantities such as stress and strain. Our method combines optical tissue clearing with second-harmonic generation imaging, Fourier-based image analysis and maximum-likelihood estimation for distribution fitting. With a new sample preparation method for arteries, we present, for the first time to our knowledge, a continuous three-dimensional distribution of collagen fibres throughout the entire thickness of the aortic wall, revealing novel structural and organizational insights into the three arterial layers.

Quantitative assessment of collagen fibre orientations from two-dimensional images of soft biological tissues

In this work, we outline an automated method for the extraction and quantification of material parameters characterizing collagen fibre orientations from two-dimensional images. Morphological collagen data among different length scales were obtained by combining the established methods of Fourier power spectrum analysis, wedge filtering and progressive regions of interest splitting. Our proposed method yields data from which we can determine parameters for computational modelling of soft biological tissues using fibre-reinforced constitutive models and gauge the length scales most appropriate for obtaining a physically meaningful measure of fibre orientations, which is representative of the true tissue morphology of the two-dimensional image. Specifically, we focus on three parameters quantifying different aspects of the collagen morphology: first, using maximum-likelihood estimation, we extract location parameters that accurately determine the angle of the principal directions of the fibre reinforcement (i.e. the preferred fibre directions); second, using a dispersion model, we obtain dispersion parameters quantifying the collagen fibre dispersion about these principal directions; third, we calculate the weighted error entropy as a measure of changes in the entire fibre distributions at different length scales, as opposed to their average behaviour. With fully automated imaging techniques (such as multiphoton microscopy) becoming increasingly popular (which often yield large numbers of images to analyse), our method provides an ideal tool for quickly extracting mechanically relevant tissue parameters which have implications for computational modelling (e.g. on the mesh density) and can also be used for the inhomogeneous modelling of tissues.

Gender Differences in Biomechanical Properties, Thrombus Age, Mass Fraction and Clinical Factors of Abdominal Aortic Aneurysms

OBJECTIVE The main purpose of the present study is the investigation of gender differences in the biomechanical properties, thrombus age, mass fraction and key clinical factors of abdominal aortic aneurysms (AAAs). MATERIALS AND METHODS A total of 90 AAA samples (78 males and 12 females) were harvested from open surgical aneurysm repairs. Biaxial extension and peeling tests were performed to characterise the biaxial mechanical responses and to determine dissection properties of both the intraluminal thrombi (ILTs) and the thrombus-covered walls. Relative thrombus age was determined by characterising the ILT histological microstructure. Mass fraction analyses quantified dry weight percentages of elastin and collagen within the AAA walls. Moreover, we statistically compared clinical factors between male and female. RESULTS The luminal layers of the female thrombi and the female AAA walls showed a significantly lower tissue stiffness (modulus) in the longitudinal direction when compared to males. Gender differences were also shown in the dissection properties of the intima-media composite within the AAA walls, in which a statistically significantly lower energy to propagate a dissection was quantified for females than for males. Moreover, 82% of female thrombi were relatively older (ILT age phases III and IV), twice that of male thrombi (43%). A pronounced lower elastin content was identified for the intima-media composites of male AAA walls, whereas female AAA walls had significantly lower dry weight percentages of collagen. Regarding clinical factors, nicotine pack years, serum creatinine and AAA expansion rate were found to be much higher for male patients. CONCLUSION These findings may help to explain higher risks for AAA growth in males and the ruptures of smaller-sized AAAs in females.

Multiaxial mechanical response and constitutive modeling of esophageal tissues: Impact on esophageal tissue engineering.

Congenital defects of the esophagus are relatively frequent, with 1 out of 2500 babies suffering from such a defect. A new method of treatment by implanting tissue engineered esophagi into newborns is currently being developed and tested using ovine esophagi. For the reconstruction of the biological function of native tissues with engineered esophagi, their cellular structure as well as their mechanical properties must be considered. Since very limited mechanical and structural data for the esophagus are available, the aim of this study was to investigate the multiaxial mechanical behavior of the ovine esophagus and the underlying microstructure. Therefore, uniaxial tensile, biaxial tensile and extension-inflation tests on esophagi were performed. The underlying microstructure was examined in stained histological sections through standard optical microscopy techniques. Moreover, the uniaxial ultimate tensile strength and residual deformations of the tissue were determined. Both the mucosa-submucosa and the muscle layers showed nonlinear and anisotropic mechanical behavior during uniaxial, biaxial and inflation testing. Cyclical inflation of the intact esophageal tube caused marked softening of the passive esophagi in the circumferential direction. The rupture strength of the mucosa-submucosa layer was much higher than that of the muscle layer. Overall, the ovine esophagus showed a heterogeneous and anisotropic behavior with different mechanical properties for the individual layers. The intact and layer-specific multiaxial properties were characterized using a well-known three-dimensional microstructurally based strain-energy function. This novel and complete set of data serves the basis for a better understanding of tissue remodeling in diseased esophagi and can be used to perform computer simulations of surgical interventions or medical-device applications.

Remodeling of Intramural Thrombus and Collagen in an Ang-II Infusion ApoE−/− Model of Dissecting Aortic Aneurysms

Fibrillar collagen endows the normal aortic wall with significant stiffness and strength and similarly plays important roles in many disease processes. For example, because of the marked loss of elastic fibers and functional smooth cells in aortic aneurysms, collagen plays a particularly important role in controlling the dilatation of these lesions and governing their rupture potential. Recent findings suggest further that collagen remodeling may also be fundamental to the intramural healing of arterial or aneurysmal dissections. To explore this possibility further, we identified and correlated regions of intramural thrombus and newly synthesized fibrillar collagen in a well-established mouse model of dissecting aortic aneurysms. Our findings suggest that intramural thrombus that is isolated from free-flowing blood creates a permissive environment for the synthesis of fibrillar collagen that, albeit initially less dense and organized, could protect that region of the dissected wall from subsequent expansion of the dissection or rupture. Moreover, alpha-smooth muscle actin positive cells appeared to be responsible for the newly produced collagen, which co-localized with significant production of glycosaminoglycans.

Selective enzymatic removal of elastin and collagen from human abdominal aortas: Uniaxial mechanical response and constitutive modeling

The ability to selectively remove the structurally most relevant components of arterial wall tissues such as collagen and elastin enables ex vivo biomechanical testing of the remaining tissues, with the aim of assessing their individual mechanical contributions. Resulting passive material parameters can be utilized in mathematical models of the cardiovascular system. Using eighteen wall specimens from non-atherosclerotic human abdominal aortas (55 ± 11 years; 9 female, 9 male), we tested enzymatic approaches for the selective digestion of collagen and elastin, focusing on their application to human abdominal aortic wall tissues from different patients with varying sample morphologies. The study resulted in an improved protocol for elastin removal, showing how the enzymatic process is affected by inadequate addition of trypsin inhibitor. We applied the resulting protocol to circumferential and axial specimens from the media and the adventitia, and performed cyclic uniaxial extension tests in the physiological and supra-physiological loading domain. The collagenase-treated samples showed a (linear) response without distinct softening behavior, while the elastase-treated samples exhibited a nonlinear, anisotropic response with pronounced remanent deformations (continuous softening), presumably caused by some sliding of collagen fibers within the damaged regions of the collagen network. In addition, our data showed that the stiffness in the initial linear stress-stretch regime at low loads is lower in elastin-free tissue compared to control samples (i.e. collagen uncrimping requires less force than the stretching of elastin), experimentally confirming that elastin is responsible for the initial stiffness in elastic arteries. Utilizing a continuum mechanical description to mathematically capture the experimental results we concluded that the inclusion of a damage model for the non-collagenous matrix material is, in general, not necessary. To model the softening behavior, continuous damage was included in the fibers by adding a damage variable which led to remanent strains through the consideration of damage.

Variations of dissection properties and mass fractions with thrombus age in human abdominal aortic aneurysms

INTRODUCTION Thrombus ages, defined as four relative age phases, are related to different compositions of the intraluminal thrombus (ILT) in the abdominal aortic aneurysm (AAA) (Tong et al., 2011b). Experimental studies indicate a correlation between the relative thrombus age and the strength of the thrombus-covered wall. METHODS On 32 AAA samples we performed peeling tests with the aim to dissect the material (i) through the ILT thickness, (ii) within the individual ILT layers and (iii) within the aneurysm wall underneath the thrombus by using two extension rates (1mm/min, 1mm/s). Histological investigations and mass fraction analysis were performed to characterize the dissected morphology, to determine the relative thrombus age, and to quantify dry weight percentages of elastin and collagen in the AAA wall. RESULTS A remarkably lower dissection energy was needed to dissect within the individual ILT layers and through the thicknesses of old thrombi. With increasing ILT age the dissection energy of the underlying intima-media composite continuously decreased and the anisotropic dissection properties for that composite vanished. The quantified dissection properties were rate dependent for both tissue types (ILT and wall). Histology showed that single fibrin fibers or smaller protein clots within the ILT generate smooth dissected surfaces during the peeling. There was a notable decrease in mass fraction of elastin within the thrombus-covered intima-media composite with ILT age, whereas no significant change was found for that of collagen. CONCLUSIONS These findings suggest that intraluminal thrombus aging leads to a higher propensity of dissection for the ILT and the intima-media composite of the aneurysmal wall.