Esther Melo

Inhomogeneity of local stiffness in the extracellular matrix scaffold of fibrotic mouse lungs.

Lung disease models are useful to study how cell engraftment, proliferation and differentiation are modulated in lung bioengineering. The aim of this work was to characterize the local stiffness of decellularized lungs in aged and fibrotic mice. Mice (2- and 24-month old; 14 of each) with lung fibrosis (N=20) and healthy controls (N=8) were euthanized after 11 days of intratracheal bleomycin (fibrosis) or saline (controls) infusion. The lungs were excised, decellularized by a conventional detergent-based (sodium-dodecyl sulfate) procedure and slices of the acellular lungs were prepared to measure the local stiffness by means of atomic force microscopy. The local stiffness of the different sites in acellular fibrotic lungs was very inhomogeneous within the lung and increased according to the degree of the structural fibrotic lesion. Local stiffness of the acellular lungs did not show statistically significant differences caused by age. The group of mice most affected by fibrosis exhibited local stiffness that were ~2-fold higher than in the control mice: from 27.2±1.64 to 64.8±7.1kPa in the alveolar septa, from 56.6±4.6 to 99.9±11.7kPa in the visceral pleura, from 41.1±8.0 to 105.2±13.6kPa in the tunica adventitia, and from 79.3±7.2 to 146.6±28.8kPa in the tunica intima. Since acellular lungs from mice with bleomycin-induced fibrosis present considerable micromechanical inhomogeneity, this model can be a useful tool to better investigate how different degrees of extracellular matrix lesion modulate cell fate in the process of organ bioengineering from decellularized lungs.

Heterogeneous micromechanical properties of the extracellular matrix in healthy and infarcted hearts

Infarcted hearts are macroscopically stiffer than healthy organs. Nevertheless, although cell behavior is mediated by the physical features of the cell niche, the intrinsic micromechanical properties of healthy and infarcted heart extracellular matrix (ECM) remain poorly characterized. Using atomic force microscopy, we studied ECM micromechanics of different histological regions of the left ventricle wall of healthy and infarcted mice. Hearts excised from healthy (n=8) and infarcted mice (n=8) were decellularized with sodium dodecyl sulfate and cut into 12 μm thick slices. Healthy ventricular ECM revealed marked mechanical heterogeneity across histological regions of the ventricular wall with the effective Youngs modulus ranging from 30.2 ± 2.8 to 74.5 ± 8.7 kPa in collagen- and elastin-rich regions of the myocardium, respectively. Infarcted ECM showed a predominant collagen composition and was 3-fold stiffer than collagen-rich regions of the healthy myocardium. ECM of both healthy and infarcted hearts exhibited a solid-like viscoelastic behavior that conforms to two power-law rheology. Knowledge of intrinsic micromechanical properties of the ECM at the length scale at which cells sense their environment will provide further insight into the cell-scaffold interplay in healthy and infarcted hearts.

Mechanical properties of acellular mouse lungs after sterilization by gamma irradiation

Lung bioengineering using decellularized organ scaffolds is a potential alternative for lung transplantation. Clinical application will require donor scaffold sterilization. As gamma-irradiation is a conventional method for sterilizing tissue preparations for clinical application, the aim of this study was to evaluate the effects of lung scaffold sterilization by gamma irradiation on the mechanical properties of the acellular lung when subjected to the artificial ventilation maneuvers typical within bioreactors. Twenty-six mouse lungs were decellularized by a sodium dodecyl sulfate detergent protocol. Eight lungs were used as controls and 18 of them were submitted to a 31kGy gamma irradiation sterilization process (9 kept frozen in dry ice and 9 at room temperature). Mechanical properties of acellular lungs were measured before and after irradiation. Lung resistance (RL) and elastance (EL) were computed by linear regression fitting of recorded signals during mechanical ventilation (tracheal pressure, flow and volume). Static (Est) and dynamic (Edyn) elastances were obtained by the end-inspiratory occlusion method. After irradiation lungs presented higher values of resistance and elastance than before irradiation: RL increased by 41.1% (room temperature irradiation) and 32.8% (frozen irradiation) and EL increased by 41.8% (room temperature irradiation) and 31.8% (frozen irradiation). Similar increases were induced by irradiation in Est and Edyn. Scanning electron microscopy showed slight structural changes after irradiation, particularly those kept frozen. Sterilization by gamma irradiation at a conventional dose to ensure sterilization modifies acellular lung mechanics, with potential implications for lung bioengineering.

Mechanical properties of mouse lungs along organ decellularization by sodium dodecyl sulfate

Lung decellularization is based on the use of physical, chemical, or enzymatic methods to break down the integrity of the cells followed by a treatment to extract the cellular material from the lung scaffold. The aim of this study was to characterize the mechanical changes throughout the different steps of lung decellularization process. Four lungs from mice (C57BL/6) were decellularized by using a conventional protocol based on sodium dodecyl sulfate. Lungs resistance (R(L)) and elastance (E(L)) were measured along decellularization steps and were computed by linear regression fitting of tracheal pressure, flow, and volume during mechanical ventilation. Transients differences found were more distinct in an intermediate step after the lungs were rinsed with deionized water and treated with 1% SDS, whereupon the percentage of variation reached approximately 80% for resistance values and 30% for elastance values. In conclusion, although a variation in extracellular matrix stiffness was observed during the decellularization process, this variation can be considered negligible overall because the resistance and elastance returned to basal values at the final decellularization step.

Development of a Bronchial Wall Model: Triple Culture on a Decellularized Porcine Trachea.

In vitro coculture models mimicking the bronchial barrier are a significant step forward in investigating the behavior and function of the upper respiratory tract mucosa. To date, mostly synthetic materials have been used as substrates to culture the cells. However, decellularized tissues provide a more in vivo-like environment based on the native extracellular matrix. In this study, an in vitro, bronchial wall coculture model has been established using a decellularized, porcine luminal trachea membrane and employing three relevant human cell types. The tissue was decellularized and placed in plastic transwell supports. The human bronchial epithelial cell line, 16HBE14o-, was seeded on the apical side of the membrane with the human lung fibroblast cell line, Wi-38, and/or the microvascular endothelial cell line, ISO-HAS-1, seeded on the basolateral side. Transepithelial electrical resistance (TER) was measured over 10 days and tight/adherens junctions (ZO-1, occludin/β-catenin) were studied through immunofluorescence. Scanning electron microscopy (SEM) was performed to evaluate microvilli and cilia formation. All cultures grew successfully on the membrane. TER values of 555 Ω·cm(2) (±21, SEM) were achieved in the monoculture. Cocultures with fibroblasts reached 565 Ω·cm(2) (±41, SEM), with endothelial cells at 638 Ω·cm(2) (±37, SEM), and the triple culture achieved 552 Ω·cm(2) (±38, SEM). ZO-1, occludin, and β-catenin were expressed in 16HBE14o- under all culture conditions. Using SEM, a dense microvilli population was found. Prominent cell-cell contacts and clusters of emerging cilia could be identified. Fibroblasts and endothelial cells strengthened the formation of a tight barrier by the 16HBE14o-. Thus, the coculture of three relevant cell types in combination with native decellularized scaffolds as a substrate approaches more closely the in vivo situation and could be used to study mechanisms of upper respiratory damage and regeneration.

Antioxidant effect of human adult adipose-derived stromal stem cells in alveolar epithelial cells undergoing stretch

INTRODUCTION Alveolar epithelial cells undergo stretching during mechanical ventilation. Stretch can modify the oxidative balance in the alveolar epithelium. The aim of the present study was to evaluate the antioxidant role of human adult adipose tissue-derived stromal cells (hADSCs) when human alveolar epithelial cells were subjected to injurious cyclic overstretching. METHODS A549 cells were subjected to biaxial stretch (0-15% change in surface area for 24h, 0.2Hz) with and without hADSCs. At the end of the experiments, oxidative stress was measured as superoxide generation using positive nuclear dihydroethidium (DHE) staining, superoxide dismutase (SOD) activity in cell lysates, 8-isoprostane concentrations in supernatant, and 3-nitrotyrosine by indirect immunofluorescence in fixed cells. RESULTS Cyclically stretching of AECs induced a significant decrease in SOD activity, and an increase in 8-isoprostane concentrations, DHE staining and 3-nitrotyrosine staining compared with non-stretched cells. Treatment with hADSCs significantly attenuated stretch-induced changes in SOD activity, 8-isoprostane concentrations, DHE and 3-nitrotyrosine staining. CONCLUSION These data suggest that hADSCs have an anti-oxidative effect in human alveolar epithelial cells undergoing cyclic stretch.