Effects of aging on the biomechanical properties of the lung extracellular matrix: dependence on tissular stretch.

Introduction: Aging induces functional and structural changes in the lung, characterized by a decline in elasticity and diminished pulmonary remodeling and regenerative capacity. Emerging evidence suggests that most biomechanical alterations in the lung result from changes in the composition of the lung extracellular matrix (ECM), potentially modulating the behavior of pulmonary cells and increasing the susceptibility to chronic lung diseases. Therefore, it is crucial to investigate the mechanical properties of the aged lung. This study aims to assess the mechanical alterations in the lung ECM due to aging at both residual (RV) and functional (FV) lung volumes and to evaluate their effects on the survival and proliferation of mesenchymal stromal cells (MSCs). Methods: The lungs from young (4-6-month-old) and aged (20-24-month-old) mice were inflated with optimal cutting temperature compound to reach FV or non-inflated (RV). ECM proteins laminin, collagen I and fibronectin were quantified by immunofluorescence and the mechanical properties of the decellularized lung sections were assessed using atomic force microscopy. To investigate whether changes in ECM composition by aging and/or mechanical properties at RV and FV volumes affects MSCs, their viability and proliferation were evaluated after 72 h. Results: Laminin presence was significantly reduced in aged mice compared to young mice, while fibronectin and collagen I were significantly increased in aged mice. In RV conditions, the acellular lungs from aged mice were significantly softer than from young mice. By contrast, in FV conditions, the aged lung ECM becomes stiffer than that of in young mice, revealing that strain hardening significantly depends on aging. Results after MSCs recellularization showed similar viability and proliferation rate in all conditions. Discussion: This data strongly suggests that biomechanical measurements, especially in aging models, should be carried out in physiomimetic conditions rather than following the conventional non-inflated lung (RV) approach. The use of decellularized lung scaffolds from aged and/or other lung disease murine/human models at physiomimetic conditions will help to better understand the potential role of mechanotransduction on the susceptibility and progression of chronic lung diseases, lung regeneration and cancer.

Cell-Laden 3D Hydrogels of Type I Collagen Incorporating Bacterial Nanocellulose Fibers.

There is a growing interest in developing natural hydrogel-based scaffolds to culture cells in a three-dimensional (3D) millieu that better mimics the in vivo cells' microenvironment. A promising approach is to use hydrogels from animal tissues, such as decellularized extracellular matrices; however, they usually exhibit suboptimal mechanical properties compared to native tissue and their composition with hundreds of different protein complicates to elucidate which stimulus triggers cell's responses. As simpler scaffolds, type I collagen hydrogels are used to study cell behavior in mechanobiology even though they are also softer than native tissues. In this work, type I collagen is mixed with bacterial nanocellulose fibers (BCf) to develop reinforced scaffolds with mechanical properties suitable for 3D cell culture. BCf were produced from blended pellicles biosynthesized from Komagataeibacter xylinus. Then, BCf were mixed with concentrated collagen from rat-tail tendons to form composite hydrogels. Confocal laser scanning microscopy and scanning electron microscopy images confirmed the homogeneous macro- and microdistribution of both natural polymers. Porosity analysis confirmed that BCf do not disrupt the scaffold structure. Tensile strength and rheology measurements demonstrated the reinforcement action of BCf (43% increased stiffness) compared to the collagen hydrogel while maintaining the same viscoelastic response. Additionally, this reinforcement of collagen hydrogels with BCf offers the possibility to mix cells before gelation and then proceed to the culture of the 3D cell scaffolds. We obtained scaffolds with human bone marrow-derived mesenchymal stromal cells or human fibroblasts within the composite hydrogels, allowing a homogeneous 3D viable culture for at least 7 days. A smaller surface shrinkage in the reinforced hydrogels compared to type I collagen hydrogels confirmed the strengthening of the composite hydrogels. These collagen hydrogels reinforced with BCf might emerge as a promising platform for 3D in vitro organ modeling, tissue-engineering applications, and suitable to conduct fundamental mechanobiology studies.

Characterization of Bioinks Prepared via Gelifying Extracellular Matrix from Decellularized Porcine Myocardia.

Since the emergence of 3D bioprinting technology, both synthetic and natural materials have been used to develop bioinks for producing cell-laden cardiac grafts. To this end, extracellular-matrix (ECM)-derived hydrogels can be used to develop scaffolds that closely mimic the complex 3D environments for cell culture. This study presents a novel cardiac bioink based on hydrogels exclusively derived from decellularized porcine myocardium loaded with human-bone-marrow-derived mesenchymal stromal cells. Hence, the hydrogel can be used to develop cell-laden cardiac patches without the need to add other biomaterials or use additional crosslinkers. The scaffold ultrastructure and mechanical properties of the bioink were characterized to optimize its production, specifically focusing on the matrix enzymatic digestion time. The cells were cultured in 3D within the developed hydrogels to assess their response. The results indicate that the hydrogels fostered inter-cell and cell-matrix crosstalk after 1 week of culture. In conclusion, the bioink developed and presented in this study holds great potential for developing cell-laden customized patches for cardiac repair.

Characterization of 3D Printed Metal-PLA Composite Scaffolds for Biomedical Applications.

Three-dimensional printing is revolutionizing the development of scaffolds due to their rapid-prototyping characteristics. One of the most used techniques is fused filament fabrication (FFF), which is fast and compatible with a wide range of polymers, such as PolyLactic Acid (PLA). Mechanical properties of the 3D printed polymeric scaffolds are often weak for certain applications. A potential solution is the development of composite materials. In the present work, metal-PLA composites have been tested as a material for 3D printing scaffolds. Three different materials were tested: copper-filled PLA, bronze-filled PLA, and steel-filled PLA. Disk-shaped samples were printed with linear infill patterns and line spacing of 0.6, 0.7, and 0.8 mm, respectively. The porosity of the samples was measured from cross-sectional images. Biocompatibility was assessed by culturing Human Bone Marrow-Derived Mesenchymal Stromal on the surface of the printed scaffolds. The results showed that, for identical line spacing value, the highest porosity corresponded to bronze-filled material and the lowest one to steel-filled material. Steel-filled PLA polymers showed good cytocompatibility without the need to coat the material with biomolecules. Moreover, human bone marrow-derived mesenchymal stromal cells differentiated towards osteoblasts when cultured on top of the developed scaffolds. Therefore, it can be concluded that steel-filled PLA bioprinted parts are valid scaffolds for bone tissue engineering.

Involvement of Mechanical Cues in the Migration of Cajal-Retzius Cells in the Marginal Zone During Neocortical Development.

Emerging evidence points to coordinated action of chemical and mechanical cues during brain development. At early stages of neocortical development, angiogenic factors and chemokines such as CXCL12, ephrins, and semaphorins assume crucial roles in orchestrating neuronal migration and axon elongation of postmitotic neurons. Here we explore the intrinsic mechanical properties of the developing marginal zone of the pallium in the migratory pathways and brain distribution of the pioneer Cajal-Retzius cells. These neurons are generated in several proliferative regions in the developing brain (e.g., the cortical hem and the pallial subpallial boundary) and migrate tangentially in the preplate/marginal zone covering the upper portion of the developing cortex. These cells play crucial roles in correct neocortical layer formation by secreting several molecules such as Reelin. Our results indicate that the motogenic properties of Cajal-Retzius cells and their perinatal distribution in the marginal zone are modulated by both chemical and mechanical factors, by the specific mechanical properties of Cajal-Retzius cells, and by the differential stiffness of the migratory routes. Indeed, cells originating in the cortical hem display higher migratory capacities than those generated in the pallial subpallial boundary which may be involved in the differential distribution of these cells in the dorsal-lateral axis in the developing marginal zone.

Mechanical ventilation promotes lung tumour spread by modulation of cholesterol cell content.

BACKGROUND: Mechanical stretch of cancer cells can alter their invasiveness. During mechanical ventilation, lungs may be exposed to an increased amount of stretch, but the consequences on lung tumours have not been explored. METHODS: To characterise the influence of mechanical ventilation on the behaviour of lung tumours, invasiveness assays and transcriptomic analyses were performed in cancer cell lines cultured in static conditions or under cyclic stretch. Mice harbouring lung melanoma implants were submitted to mechanical ventilation and metastatic spread was assessed. Additional in vivo experiments were performed to determine the mechanodependent specificity of the response. Incidence of metastases was studied in a cohort of lung cancer patients that received mechanical ventilation compared with a matched group of nonventilated patients. RESULTS: Stretch increases invasiveness in melanoma B16F10luc2 and lung adenocarcinoma A549 cells. We identified a mechanosensitive upregulation of pathways involved in cholesterol processing in vitro, leading to an increase in pro-protein convertase subtilisin/kexin type 9 (PCSK9) and LDLR expression, a decrease in intracellular cholesterol and preservation of cell stiffness. A course of mechanical ventilation in mice harbouring melanoma implants increased brain and kidney metastases 2 weeks later. Blockade of PCSK9 using a monoclonal antibody increased cell cholesterol and stiffness and decreased cell invasiveness in vitro and metastasis in vivo. In patients, mechanical ventilation increased PCSK9 abundance in lung tumours and the incidence of metastasis, thus decreasing survival. CONCLUSIONS: Our results suggest that mechanical stretch promote invasiveness of cancer cells, which may have clinically relevant consequences. Pharmacological manipulation of cholesterol endocytosis could be a novel therapeutic target in this setting.

Experimental Setting for Applying Mechanical Stimuli to Study the Endothelial Response of Ex Vivo Vessels under Realistic Pathophysiological Environments.

This paper describes the design, construction and testing of an experimental setting, making it possible to study the endothelium under different pathophysiological conditions. This novel experimental approach allows the application of the following stimuli to an ex vivo vessel in a physiological bath: (a) a realistic intravascular pressure waveform defined by the user; (b) shear stress in the endothelial layer since, in addition to the pressure waveform, the flow through the vessel can be independently controlled by the user; (c) conditions of hypo/hyperoxia and hypo/hypercapnia in an intravascular circulating medium. These stimuli can be applied alone or in different combinations to study possible synergistic or antagonistic effects. The setting performance is illustrated by a proof of concept in an ex vivo rabbit aorta. The experimental setting is easy to build by using very low-cost materials widely available. Online Supplement files provide all the technical information (e.g., circuits, codes, 3D printer drivers) following an open-source hardware approach for free replication.

Bioprintable Lung Extracellular Matrix Hydrogel Scaffolds for 3D Culture of Mesenchymal Stromal Cells.

Mesenchymal stromal cell (MSC)-based cell therapy in acute respiratory diseases is based on MSC secretion of paracrine factors. Several strategies have proposed to improve this are being explored including pre-conditioning the MSCs prior to administration. We here propose a strategy for improving the therapeutic efficacy of MSCs based on cell preconditioning by growing them in native extracellular matrix (ECM) derived from the lung. To this end, a bioink with tunable stiffness based on decellularized porcine lung ECM hydrogels was developed and characterized. The bioink was suitable for 3D culturing of lung-resident MSCs without the need for additional chemical or physical crosslinking. MSCs showed good viability, and contraction assays showed the existence of cell-matrix interactions in the bioprinted scaffolds. Adhesion capacity and length of the focal adhesions formed were increased for the cells cultured within the lung hydrogel scaffolds. Also, there was more than a 20-fold increase of the expression of the CXCR4 receptor in the 3D-cultured cells compared to the cells cultured in plastic. Secretion of cytokines when cultured in an in vitro model of lung injury showed a decreased secretion of pro-inflammatory mediators for the cells cultured in the 3D scaffolds. Moreover, the morphology of the harvested cells was markedly different with respect to conventionally (2D) cultured MSCs. In conclusion, the developed bioink can be used to bioprint structures aimed to improve preconditioning MSCs for therapeutic purposes.

Silk-Reinforced Collagen Hydrogels with Raised Multiscale Stiffness for Mesenchymal Cells 3D Culture.

Type I collagen hydrogels are of high interest in tissue engineering. With the evolution of 3D bioprinting technologies, a high number of collagen-based scaffolds have been reported for the development of 3D cell cultures. A recent proposal was to mix collagen with silk fibroin derived from Bombyx mori silkworm. Nevertheless, due to the difficulties in the preparation and the characteristics of the protein, several problems such as phase separation and collagen denaturation appear during the procedure. Therefore, the common solution is to diminish the concentration of collagen although in that way the most biologically relevant component is reduced. In this study, we present a new, simple, and effective method to develop a collagen-silk hybrid hydrogel with high collagen concentration and with increased stiffness approaching that of natural tissues, which could be of high interest for the development of cardiac patches for myocardial regeneration and for preconditioning of mesenchymal stem cells (MSCs) to improve their therapeutic potential. Sericin in the silk was preserved by using a physical solubilizing procedure that results in a preserved fibrous structure of type I collagen, as shown by ultrastructural imaging. The macro- and micromechanical properties of the hybrid hydrogels measured by tensile stretch and atomic force microscopy, respectively, showed a more than twofold stiffening than the collagen-only hydrogels. Rheological measurements showed improved printability properties for the developed biomaterial. The suitability of the hydrogels for 3D cell culture was assessed by 3D bioprinting bone marrow-derived MSCs cultured within the scaffolds. The result was a biomaterial with improved printability characteristics that better resembled the mechanical properties of natural soft tissues while preserving biocompatibility owing to the high concentration of collagen. Impact statement In this study, we report the development of silk microfiber-reinforced type I collagen hydrogels for 3D bioprinting and cell culture. In contrast with previously reported studies, a novel physical method allowed the preservation of the silk sericin protein. Hydrogels were stable, showed no phase separation between the biomaterials, and they presented improved printability. An increase between two- and threefold of the multiscale stiffness of the scaffolds was achieved with no need of using additional crosslinkers or complex methods, which could be of high relevance for cardiac patches development and for preconditioning mesenchymal stem cells (MSCs) for therapeutic applications. We demonstrate that bone marrow-derived MSCs can be effectively bioprinted and 3D cultured within the stiffened structures.