Ming-Jay Chow

Arterial Extracellular Matrix: A Mechanobiological Study of the Contributions and Interactions of Elastin and Collagen

The complex network structure of elastin and collagen extracellular matrix (ECM) forms the primary load bearing components in the arterial wall. The structural and mechanobiological interactions between elastin and collagen are important for properly functioning arteries. Here, we examined the elastin and collagen organization, realignment, and recruitment by coupling mechanical loading and multiphoton imaging. Two-photon excitation fluorescence and second harmonic generation methods were performed with a multiphoton video-rate microscope to capture real time changes to the elastin and collagen structure during biaxial deformation. Enzymatic removal of elastin was performed to assess the structural changes of the remaining collagen structure. Quantitative analysis of the structural changes to elastin and collagen was made using a combination of two-dimensional fast Fourier transform and fractal analysis, which allows for a more complete understanding of structural changes. Our study provides new quantitative evidence, to our knowledge on the sequential engagement of different arterial ECM components in response to mechanical loading. The adventitial collagen exists as large wavy bundles of fibers that exhibit fiber engagement after 20% strain. The medial collagen is engaged throughout the stretching process, and prominent elastic fiber engagement is observed up to 20% strain after which the engagement plateaus. The fiber orientation distribution functions show remarkably different changes in the ECM structure in response to mechanical loading. The medial collagen shows an evident preferred circumferential distribution, however the fiber families of adventitial collagen are obscured by their waviness at no or low mechanical strains. Collagen fibers in both layers exhibit significant realignment in response to unequal biaxial loading. The elastic fibers are much more uniformly distributed and remained relatively unchanged due to loading. Removal of elastin produces similar structural changes in collagen as mechanical loading. Our study suggests that the elastic fibers are under tension and impart an intrinsic compressive stress on the collagen.

Experimental and Modeling Study of Collagen Scaffolds with the Effects of Crosslinking and Fiber Alignment

Collagen type I scaffolds are commonly used due to its abundance, biocompatibility, and ubiquity. Most applications require the scaffolds to operate under mechanical stresses. Therefore understanding and being able to control the structural-functional integrity of collagen scaffolds becomes crucial. Using a combined experimental and modeling approach, we studied the structure and function of Type I collagen gel with the effects of spatial fiber alignment and crosslinking. Aligned collagen scaffolds were created through the flow of magnetic particles enmeshed in collagen fibrils to mimic the anisotropy seen in native tissue. Inter- and intra- molecular crosslinking was modified chemically with Genipin to further improve the stiffness of collagen scaffolds. The anisotropic mechanical properties of collagen scaffolds were characterized using a planar biaxial tensile tester and parallel plate rheometer. The tangent stiffness from biaxial tensile test is two to three orders of magnitude higher than the storage moduli from rheological measurements. The biphasic nature of collagen gel was discussed and used to explain the mechanical behavior of collagen scaffolds under different types of mechanical tests. An anisotropic hyperelastic constitutive model was used to capture the characteristics of the stress-strain behavior exhibited by collagen scaffolds.

Characterization of biaxial mechanical behavior of porcine aorta under gradual elastin degradation.

Arteries are composed of multiple constituents that endow the wall with proper structure and function. Many vascular diseases are associated with prominent mechanical and biological alterations in the wall constituents. In this study, planar biaxial tensile test data of elastase-treated porcine aortic tissue (Chow et al. in Biomech Model Mechanobiol 2013) is re-examined to characterize the altered mechanical behavior at multiple stages of digestion through constitutive modeling. Exponential-based as well as recruitment-based strain energy functions are employed and the associated constitutive parameters for individual digestion stages are identified using nonlinear parameter estimation. It is shown that when the major portion of elastin is degraded from a cut-open artery in the load-free state, the embedded collagen fibers are recruited at lower stretch levels under biaxial loads, leading to a rapid stiffening behavior of the tissue. Multiphoton microscopy illustrates that the collagen waviness decreases significantly with the degradation time, resulting in a rapid recruitment when the tissue is loaded. It is concluded that even when residual stresses are released, there exists an intrinsic mechanical interaction between arterial elastin and collagen that determines the mechanics of arteries and carries important implications to vascular mechanobiology.

Contribution of Collagen Fiber Undulation to Regional Biomechanical Properties Along Porcine Thoracic Aorta

As major extracellular matrix components, elastin, and collagen play crucial roles in regulating the mechanical properties of the aortic wall and, thus, the normal cardiovascular function. The mechanical properties of aorta, known to vary with age and multitude of diseases as well as the proximity to the heart, have been attributed to the variations in the content and architecture of wall constituents. This study is focused on the role of layer-specific collagen undulation in the variation of mechanical properties along the porcine descending thoracic aorta. Planar biaxial tensile tests are performed to characterize the hyperelastic anisotropic mechanical behavior of tissues dissected from four locations along the thoracic aorta. Multiphoton microscopy is used to image the associated regional microstructure. Exponential-based and recruitment-based constitutive models are used to account for the observed mechanical behavior while considering the aortic wall as a composite of two layers with independent properties. An elevated stiffness is observed in distal regions compared to proximal regions of thoracic aorta, consistent with sharper and earlier collagen recruitment estimated for medial and adventitial layers in the models. Multiphoton images further support our prediction that higher stiffness in distal regions is associated with less undulation in collagen fibers. Recruitment-based models further reveal that regardless of the location, collagen in the media is recruited from the onset of stretching, whereas adventitial collagen starts to engage with a delay. A parameter sensitivity analysis is performed to discriminate between the models in terms of the confidence in the estimated model parameters.

Progressive structural and biomechanical changes in elastin degraded aorta

Aortic aneurysm is an important clinical condition characterized by common structural changes such as the degradation of elastin, loss of smooth muscle cells, and increased deposition of fibrillary collagen. With the goal of investigating the relationship between the mechanical behavior and the structural/biochemical composition of an artery, this study used a simple chemical degradation model of aneurysm and investigated the progressive changes in mechanical properties. Porcine thoracic aortas were digested in a mild solution of purified elastase (5 U/mL) for 6, 12, 24, 48, and 96 h. Initial size measurements show that disruption of the elastin structure leads to increased artery dilation in the absence of periodic loading. The mechanical properties of the digested arteries, measured with a biaxial tensile testing device, progress through four distinct stages termed (1) initial-softening, (2) elastomer-like, (3) extensible-but-stiff, and (4) collagen-scaffold-like. While stages 1, 3, and 4 are expected as a result of elastin degradation, the S-shaped stress versus strain behavior of the aorta resulting from enzyme digestion has not been reported previously. Our results suggest that gradual changes in the structure of elastin in the artery can lead to a progression through different mechanical properties and thus reveal the potential existence of an important transition stage that could contribute to artery dilation during aneurysm formation.

The Effect of Static Stretch on Elastin Degradation in Arteries

Previously we have shown that gradual changes in the structure of elastin during an elastase treatment can lead to important transition stages in the mechanical behavior of arteries [1]. However, in vivo arteries are constantly being loaded due to systolic and diastolic pressures and so understanding the effects of loading on the enzymatic degradation of elastin in arteries is important. With biaxial tensile testing, we measured the mechanical behavior of porcine thoracic aortas digested with a mild solution of purified elastase (5 U/mL) in the presence of a static stretch. Arterial mechanical properties and biochemical composition were analyzed to assess the effects of mechanical stretch on elastin degradation. As elastin is being removed, the dimensions of the artery increase by more than 20% in both the longitude and circumference directions. Elastin assays indicate a faster rate of degradation when stretch was present during the digestion. A simple exponential decay fitting confirms the time constant for digestion with stretch (0.11±0.04 h−1) is almost twice that of digestion without stretch (0.069±0.028 h−1). The transition from J-shaped to S-shaped stress vs. strain behavior in the longitudinal direction generally occurs when elastin content is reduced by about 60%. Multiphoton image analysis confirms the removal/fragmentation of elastin and also shows that the collagen fibers are closely intertwined with the elastin lamellae in the medial layer. After removal of elastin, the collagen fibers are no longer constrained and become disordered. Release of amorphous elastin during the fragmentation of the lamellae layers is observed and provides insights into the process of elastin degradation. Overall this study reveals several interesting microstructural changes in the extracellular matrix that could explain the resulting mechanical behavior of arteries with elastin degradation.

Obstruction-Induced Pulmonary Vascular Remodeling

Pulmonary obstruction occurs in many common forms of congenital heart disease. In this study, pulmonary artery (PA) banding is used as a model for pulmonary stenosis. Significant remodeling of the vascular bed occurs as a result of a prolonged narrowing of the PAs, and here we quantify the biophysical and molecular changes proximal and distal to the obstruction. Main and branch PAs are harvested from banded and sham rabbits and their mechanical properties are assessed using a biaxial tensile tester. Measurements defined as initial and stiff slopes are taken, assuming a linear region at the start and end of the J-shaped stress-strain curves, along with a transitional knee point. Collagen, elastin assays, Movats pentachrome staining, and Doppler protocols are used to quantify biochemical, structural, and physiological differences. The banded main PAs have significantly greater initial slopes while banded branch PAs have lower initial slopes; however, this change in mechanical behavior cannot be explained by the assay results as the elastin content in both main and branch PAs is not significantly different. The stiff slopes of the banded main PAs are higher, which is attributed to the significantly greater amounts of insoluble collagen. Shifting of the knee points reveals a decreased toe region in the main PAs but an opposite trend in the branch PAs. The histology results show a loss of integrity of the media, increase in ground substance, and dispersion of collagen in the banded tissue samples. This indicates other structural changes could have led to the mechanical differences in banded and normal tissue.

Elastin in the Arterial ECM: Interactions With Collagen and the Mechanical Properties After Elastin Degradation

Elastin and collagen are the main structural components in the extracellular matrix (ECM) that contribute to the anisotropic and hyperelastic passive mechanical behavior of elastic arteries. It is commonly accepted that the elastin fibers support most of the load at the onset of stretching while collagen fiber recruitment and the transition to collagen bearing the load occurs at higher pressures [1]. Various diseases lead to changes in the ECM, for example in aortic aneurysm there is reduced elastin, excess aged collagen, and fragmentation of the elastic lamellae [2]. Likewise hypertension has been shown to increase arterial collagen and wall thickness with increased stiffness [3]. Improving our knowledge of how the ECM structure affects the mechanical behavior of arteries can provide insights to disease progression and better treatment methods.© 2013 ASME

Quantifying the Structural and Mechanical Changes in Elastase Degraded Arteries as an In Vitro Model of Aortic Aneurysm

Common characteristics of aortic aneurysm include loss of elastin/smooth muscle cells, increase in fibrillary collagen, and increase in artery diameter [5]. Because of the high mortality rate of aneurysm rupture, it is desirable to be able to predict when a patient should have surgery to repair the dilated tissue. Current clinical practices involve predicting aneurysm rupture based on artery expansion rate and diameter. However, other parameters such as wall stiffness and peak wall stress may offer better predictions as to when an aneurysm will fail [8]. Previous studies have investigated the differences in elastin and collagen content of abdominal aortic tissue with and without abdominal aortic aneurysm (AAA) [1]. In another study, human aortic aneurysm tissue was tested in a biaxial tensile tester and the resulting stress strain curves were fitted using Fung type exponential strain energy function [7]. More extensive modeling of aneurysm tissue has been done by modifying the Holzapfel model to incorporate a parameter that characterizes the tissue weakening before the failure of the inner elastic laminae, ground matrix, or collagen fibers themselves [6]. Previous studies have found compositional and mechanical differences between aneurysm and healthy tissue. In addition, good structurally based models for arteries that are developing aneurysm exist but these are mostly theoretical [6]. In order to improve aneurysm rupture prediction techniques, a better understanding of how structural changes affect the mechanical properties of the artery is necessary.Copyright