Decellularization of xenogeneic hearts offers an acellular, naturally occurring, 3D scaffold that may aid in the development of an engineered human heart tissue. However, decellularization impacts the structural and mechanical properties of the extracellular matrix (ECM), which can strongly influence a cell response during recellularization. We hypothesized that multiphoton microscopy (MPM), combined with image correlation spectroscopy (ICS), could be used to characterize the structural and mechanical properties of the decellularized cardiac matrix in a noninvasive and nondestructive fashion. Whole porcine hearts were decellularized for 7 days by four different solutions of Trypsin and/or Triton. The compressive modulus of the cardiac ECM decreased to < 20% of that of the native tissue in three of the four conditions (range 2-8 kPa); the modulus increased by -150% (range 125-150 kPa) in tissues treated with Triton only. The collagen and elastin content decreased steadily over time for all four decellularization conditions. The ICS amplitude of second harmonic generation (SHG, ASHG) collagen images increased in three of the four decellularization conditions characterized by a decrease in fiber density; the ICS amplitude was approximately constant in tissues treated with Triton only. The ICS ratio (R(SHG), skew) of collagen images increased significantly in the two conditions characterized by a loss of collagen crimping or undulations. The ICS ratio of two-photon fluorescence (TPF, R(TPF)) elastin images decreased in three of the four conditions, but increased significantly in Triton-only treated tissue characterized by retention of densely packed elastin fibers. There were strong linear relationships between both the log of A(SHG) (R(2) = 0.86) and R(TPF) (R(2) = 0.92) with the compressive modulus. Using these variables, a linear model predicts the compressive modulus: E=73.9 × Log(A(SHG))+70.1 × R(TPF) - 131 (R(2) = 0.94). This suggests that the collagen content and elastin alignment determine the mechanical properties of the ECM. We conclude that MPM and ICS analysis is a noninvasive, nondestructive method to predict the mechanical properties of the decellularized cardiac ECM.

Extracellular matrix (ECM) derived from whole organ decellularization has been successfully used in a variety of tissue engineering applications. ECM contains a complex mixture of functional and structural molecules that are ideally suited for the tissue from which the ECM is harvested. However, decellularization disrupts the structural properties and protein composition of the ECM, which may impact function when cells such as the fibroblast are reintroduced during recellularization. We hypothesized that the ECM structure and composition, fibroblast source, and integrin expression would influence the fibroblast phenotype. Human cardiac fibroblasts (HCFs) and normal human lung fibroblasts (NHLFs) were cultured on intact cardiac ECM, collagen gels, and coatings composed of cardiac ECM, lung ECM, and individual ECM components (collagen and fibronectin [FN]) for 48 h. COL1A expression of HCFs and NHLFs cultured on ECM and FN coatings decreased to <50% of that of untreated cells; COL1A expression for HCFs cultured on ECM coatings was one-to twofold higher than HCFs cultured on intact ECM. NHLFs cultured on ECM and FN coatings expressed 12-to 31-fold more alpha-smooth muscle actin (aSMA) than HCFs; the aSMA expression for HCFs and NHLFs cultured on ECM coatings was *2-to 5-fold higher than HCFs and NHLFs cultured on intact ECM. HCFs expressed significantly higher levels of b 3 and b 4 integrins when compared to NHLFs. Inhibition of the b 3 integrin, but not b 4 , resulted in a 16-to 26-fold increase in aSMA expression in HCFs cultured on ECM coatings and FN. Our results demonstrate that b 3 integrin expression depends on the source of the fibroblast and that its expression inhibits aSMA expression (and thus the myofibroblast phenotype). We conclude that the fibroblast source and integrin expression play important roles in regulating the fibroblast phenotype.

Our findings suggest that increased endothelial luminal surface stiffness in microvascular cells may facilitate mechanotransduction and alignment in response to laminar shear stress. Furthermore, the arachidonic acid pathway may mediate this tissue-specific process. An improved understanding of this response will aid in the treatment of organ-specific vascular disease.

Objective Preconditioning of endothelial cells from different vascular beds has potential value for re‐endothelialization and implantation of engineered tissues. Understanding how substrate stiffness and composition affects tissue‐specific cell response to shear stress will aid in successful endothelialization of engineered tissues. We developed a platform to test biomechanical and biochemical stimuli. Methods A novel polydimethylsiloxane‐based parallel plate flow chamber enabled application of laminar fluid shear stress of 2 dynes/cm2 for 12 hours to microvascular cardiac and lung endothelial cells cultured on cardiac and lung‐derived extracellular matrix. Optical imaging of cells was used to quantify cell changes in cell alignment. Analysis of integrin expression was performed using flow cytometry. Results Application of fluid shear stress caused the greatest cell alignment in cardiac endothelial cells seeded on polystyrene and lung endothelial cells on polydimethylsiloxane. This resulted in elongation of the lung endothelial cells. αv and β3 integrin expression decreased after application of shear stress in both cell types. Conclusion Substrate stiffness plays an important role in regulating tissue‐specific endothelial response to shear stress, which may be due to differences in their native microenvironments. Furthermore, cardiac and lung endothelial cell response to shear stress was significantly regulated by the type of coating used.

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