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

Abstract

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±7.1 kPa in the alveolar septa, from 56.6±4.6 to 99.9±11.7 kPa in the visceral pleura, from 41.1±8.0 to 105.2±13.6 kPa in the tunica adventitia, and from 79.3±7.2 to 146.6±28.8 kPa 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.

Introduction

Lung transplantation is the only current therapeutic indication for patients suffering the most advanced stage of devastating respiratory diseases such as chronic obstructive pulmonary disease, lung fibrosis or pulmonary arterial hypertension (Orens and Garrity, 2009). These chronic respiratory pathologies are becoming more prevalent worldwide as a result of the rise in life expectation achieved by improvements in medical treatments and public health services (Lopez et al., 2006). There is, therefore, an increasing need of viable lungs for transplantation that cannot be fully fulfilled by available donors, resulting in a progressively increasing waiting list for lung transplantation (Yusen et al., 2010).

Lung bioengineering has emerged in the recent years as a potential alternative to obtain viable organs for transplantation in the near future. In fact, proof of concept experiments of this approach have been recently reported in rodent models (Ott et al., 2010, Petersen et al., 2010). As it is common in organ bioengineering, the approach for lung biofabrication is based on the decellularization of lungs not viable for transplantation, and using their acellular matrix as a scaffold to seed stem cells –for instance induced pluripotent stem cells from the receptor to avoid organ rejection– for repopulating the whole organ after cell proliferation and differentiation (Ghaedi et al., 2013, Huang et al., 2013).

Although a considerable research effort has been recently devoted to lung bioengineering (Wagner et al., 2013, Badylak et al., 2012, Weiss, 2013), the mechanisms governing cell homing, proliferation and differentiation onto the many cell phenotypes in the lung are not well understood. However, there is reported evidence that, in addition to biochemical soluble factors, mechanical cues such as substrate stiffness and 3D structure would play a role in modulating the processes of cell repopulation within the acellular lung scaffold (Cortiella et al., 2006, Cortiella et al., 2010). Accordingly, a detailed knowledge of the mechanical properties of the acellular lung microenvironment will allow a better understanding of the mechanisms involved in cell recellularization, potentially helping to improve effectiveness of the process in lung bioengineering. Specifically, the use of acellular lungs from healthy and diseased individuals, either young or aged, is of considerable importance for investigating the potential role played by the mechanical characteristics of the extracellular matrix on cell fate during acellular lung recellularization.

In this context, aging and fibrotic mouse models have been recently suggested as research platforms for better understanding of the cell–matrix interaction in lung tissue engineering (Sokocevic et al., 2013, Booth et al., 2012). Nevertheless, these common rodent models have not been previously characterized in terms of local micromechanics. Indeed, mechanical properties of acellular lungs were studied from pressure–volume curves during inflation and deflation of the decellularized whole organ (Petersen et al., 2010). Mechanical characterization was also carried out through stress–strain curves in strips of decellularized lung tissue (Petersen et al., 2012). Nevertheless, these two approaches do not provide information at the microscale sensed by cells. Interestingly, atomic force microscopy (AFM), has proven to be a convenient tool since the method is based on a sharp tip that allows reaching nanometer resolution. By using AFM local mechanical differences have been found between different regions of the extracellular matrix in decellularized lungs (Luque et al., 2013, Melo et al., 2013) and also between acellular lung matrices belonging to healthy and diseased donors (Booth et al., 2012). However, there are no available data describing how the local stiffness of different sites of interest in the lung such as the alveolar septa, the visceral pleura or the tunicae adventitia and intima of the lung vessel walls are modified in fibrotic areas as compared with controls or whether the changes in local mechanics are uniformly or heterogeneously distributed within the lung. Therefore, the aim of this work was to measure local lung micromechanics in the acellular organs of young and aged healthy and fibrotic mice by means of AFM using pyramidal cantilever tips as mechanical probe. The spatial resolution of this tool allowed the measurement of local stiffness at the same scale that cells sense their extracellular matrix microenvironment.