Mechanical evaluation of decellularized porcine thoracic aorta.

BACKGROUND: Decellularized tissues are expected to have major cellular immunogenic components removed and in the meantime maintain similar mechanical strength and extracellular matrix (ECM) structure. However, the decellularization processes likely cause alterations of the ECM structure and thus influence the mechanical properties. In the present study, the effects of different decellularization protocols on the (passive) mechanical properties of the resulted porcine aortic ECM were evaluated. METHODS: Decellularization methods using anionic detergent (sodium dodecyl sulfate), enzymatic detergent (Trypsin), and non-ionic detergent [tert-octylphenylpolyoxyethylen (Triton X-100)] were adopted to obtain decellularized porcine aortic ECM. Histologic studies and scanning electron microscopy were performed to confirm the removal of cells and to examine the structure of ECM. Biaxial tensile testing was used to characterize both the elastic and viscoelastic mechanical behaviors of decellularized ECM. RESULTS: All three decellularization protocols remove the cells effectively. The major ECM structure is preserved under sodium dodecyle sulfate (SDS) and Triton X-100 treatments. However, the structure of Trypsin treated ECM is severely disrupted. SDS and Triton X-100 decellularized ECM exhibits similar elastic properties as intact aorta tissues. Decellularized ECM shows less stress relaxation than intact aorta due to the removal of cells. Creep behavior is negligible for both decellularized ECM and intact aortas. CONCLUSION: SDS and Triton X-100 decellularized ECM tissue appeared to maintain the critical mechanical and structural properties and might work as a potential material for further vascular tissue engineering.

Transmural variation in elastin fiber orientation distribution in the arterial wall.

The complex three-dimensional elastin network is a major load-bearing extracellular matrix (ECM) component of an artery. Despite the reported anisotropic behavior of arterial elastin network, it is usually treated as an isotropic material in constitutive models. Our recent multiphoton microscopy study reported a relatively uniform elastin fiber orientation distribution in porcine thoracic aorta when imaging from the intima side (Chow et al., 2014). However it is questionable whether the fiber orientation distribution obtained from a small depth is representative of the elastin network structure in the arterial wall, especially when developing structure-based constitutive models. To date, the structural basis for the anisotropic mechanical behavior of elastin is still not fully understood. In this study, we examined the transmural variation in elastin fiber orientation distribution in porcine thoracic aorta and its association with elastin anisotropy. Using multi-photon microscopy, we observed that the elastin fibers orientation changes from a relatively uniform distribution in regions close to the luminal surface to a more circumferential distribution in regions that dominate the media, then to a longitudinal distribution in regions close to the outer media. Planar biaxial tensile test was performed to characterize the anisotropic behavior of elastin network. A new structure-based constitutive model of elastin network was developed to incorporate the transmural variation in fiber orientation distribution. The new model well captures the anisotropic mechanical behavior of elastin network under both equi- and nonequi-biaxial loading and showed improvements in both fitting and predicting capabilities when compared to a model that only considers the fiber orientation distribution from the intima side. We submit that the transmural variation in fiber orientation distribution is important in characterizing the anisotropic mechanical behavior of elastin network and should be considered in constitutive modeling of an artery.

Micropatterned cell sheets as structural building blocks for biomimetic vascular patches.

To successfully develop a functional tissue-engineered vascular patch, recapitulating the hierarchical structure of vessel is critical to mimic mechanical properties. Here, we use a cell sheet engineering strategy with micropatterning technique to control structural organization of bovine aortic vascular smooth muscle cell (VSMC) sheets. Actin filament staining and image analysis showed clear cellular alignment of VSMC sheets cultured on patterned substrates. Viability of harvested VSMC sheets was confirmed by Live/Dead® cell viability assay after 24 and 48 h of transfer. VSMC sheets stacked to generate bilayer VSMC patches exhibited strong inter-layer bonding as shown by lap shear test. Uniaxial tensile testing of monolayer VSMC sheets and bilayer VSMC patches displayed nonlinear, anisotropic stress-stretch response similar to the biomechanical characteristic of a native arterial wall. Collagen content and structure were characterized to determine the effects of patterning and stacking on extracellular matrix of VSMC sheets. Using finite-element modeling to simulate uniaxial tensile testing of bilayer VSMC patches, we found the stress-stretch response of bilayer patterned VSMC patches under uniaxial tension to be predicted using an anisotropic hyperelastic constitutive model. Thus, our cell sheet harvesting system combined with biomechanical modeling is a promising approach to generate building blocks for tissue-engineered vascular patches with structure and mechanical behavior mimicking native tissue.

Understanding the viscoelastic behavior of collagen matrices through relaxation time distribution spectrum.

This study aims to provide understanding of the macroscopic viscoelastic behavior of collagen matrices through studying the relaxation time distribution spectrum obtained from stress relaxation tests. Hydrated collagen gel and dehydrated collagen thin film was exploited as two different hydration levels of collagen matrices. Genipin solution was used to induce crosslinking in collagen matrices. Biaxial stress relaxation tests were performed to characterize the viscoelastic behavior of collagen matrices. The rate of stress relaxation of both hydrated and dehydrated collagen matrices shows a linear initial stress level dependency. Increased crosslinking reduces viscosity in collagen gel, but the effect is negligible for thin film. Relaxation time distribution spectrum was obtained from the stress relaxation data by inverse Laplace transform. For most of the collagen matrices, three peaks at the short (0.3s ~1 s), medium (3s ~90 s), and long relaxation time (> 200 s) were observed in the continuous spectrum, which likely corresponds to relaxation mechanisms involve fiber, inter-fibril, and fibril sliding. Splitting of the middle peak was observed at higher initial stress levels suggesting increased structural heterogeneity at the fibril level with mechanical loading. The intensity of the long-term peaks increases with higher initial stress levels indicating the engagement of collagen fibrils at higher levels of tissue strain.