Publications

Microbial biofilms are complex three-dimensional structures where sessile microbes are embedded in a polymeric extracellular matrix. Their resistance toward the host immune system as well as to a diverse range of antimicrobial treatments poses a serious health and development threat, being in the top 10 global public health threats declared by the World Health Organization. In an effort to combat biofilm-related microbial infections, several strategies have been developed to independently eliminate biofilms or to complement conventional antibiotic therapies. However, their limitations leave room for other treatment alternatives, where the application of nanotechnology to biofilm eradication has gained significant relevance in recent years. Their small size, penetration efficiency, and the design flexibility that they present makes them a promising alternative for biofilm infection treatment, although they also present set-backs. This review aims to describe the main possibilities and limitations of nanomedicine against biofilms, while covering the main aspects of biofilm formation and study, and the current therapies for biofilm treatment. This article is categorized under: Therapeutic Approaches and Drug Discovery > Nanomedicine for Infectious Disease Toxicology and Regulatory Issues in Nanomedicine > Toxicology of Nanomaterials Toxicology and Regulatory Issues in Nanomedicine > Regulatory and Policy Issues in Nanomedicine.

JTD Keywords: Anti-bacterial agents, Anti-infective agents, Antiinfective agent, Antimicrobial, Antimicrobials, Antimicrobials,bacteria,biofilm,infectious diseases,microorganism, Bacteria, Biofilm, Biofilm infections, Biofilms, Complex three dimensional structures, Diseases, Diverse range, Drug-delivery systems,in-vitro,cellular toxicity,nanoparticles,penetration,model,biocompatibility,perspectives,hyperthermia,diagnosi, Extracellular matrices, Global public health, Health risks, Infectious disease, Infectious diseases, Medical nanotechnology, Microbial biofilm, Microorganisms, Nanomedicine, Polymer, Polymers, Regulatory issues, Roadmap

The performance of single-chain polymeric nanoparticles (SCPNs) in biomedical applications highly depends on their conformational stability in cellular environments. Until now, such stability studies are limited to 2D cell culture models, which do not recapitulate the 3D tumor microenvironment well. Here, a microfluidic tumor-on-a-chip model is introduced that recreates the tumor milieu and allows in-depth insights into the diffusion, cellular uptake, and stability of SCPNs. The chip contains Matrigel/collagen-hyaluronic acid as extracellular matrix (ECM) models and is seeded with cancer cell MCF7 spheroids. With this 3D platform, it is assessed how the polymer's microstructure affects the SCPN's behavior when crossing the ECM, and evaluates SCPN internalization in 3D cancer cells. A library of SCPNs varying in microstructure is prepared. All SCPNs show efficient ECM penetration but their cellular uptake/stability behavior depends on the microstructure. Glucose-based nanoparticles display the highest spheroid uptake, followed by charged nanoparticles. Charged nanoparticles possess an open conformation while nanoparticles stabilized by internal hydrogen bonding retain a folded structure inside the tumor spheroids. The 3D microfluidic tumor-on-a-chip platform is an efficient tool to elucidate the interplay between polymer microstructure and SCPN's stability, a key factor for the rational design of nanoparticles for targeted biological applications.© 2024 The Authors. Small Methods published by Wiley-VCH GmbH.

JTD Keywords: 3d cancer cell uptake, Cancer cells, Cell culture, Cell uptake, Cellular uptake, Diseases, Ecm penetration, Extracellular matrices, Extracellular matrix penetration, Functional polymers, Hydrogen bonds, Medical applications, Microfluidics, Microstructure, Nanoparticles, Polymeric nanoparticles, Scpns, Single chains, Single-chain polymeric nanoparticle, Stability, Tumor-on-a-chip, Tumors

JTD Keywords: Decellularised matrices, Nervous system (brain-central-peripheral / disorders)

Mimicking bone extracellular matrix (ECM) is paramount to develop novel biomaterials for bone tissue engineering. In this regard, the combination of integrin-binding ligands together with osteogenic peptides represents a powerful approach to recapitulate the healing microenvironment of bone. In the present work, we designed polyethylene glycol (PEG)-based hydrogels functionalized with cell instructive multifunctional biomimetic peptides (either with cyclic RGD-DWIVA or cyclic RGD-cyclic DWIVA) and cross-linked with matrix metalloproteinases (MMPs)-degradable sequences to enable dynamic enzymatic biodegradation and cell spreading and differentiation. The analysis of the intrinsic properties of the hydrogel revealed relevant mechanical properties, porosity, swelling and degradability to engineer hydrogels for bone tissue engineering. Moreover, the engineered hydrogels were able to promote human mesenchymal stem cells (MSCs) spreading and significantly improve their osteogenic differentiation. Thus, these novel hydrogels could be a promising candidate for applications in bone tissue engineering, such as acellular systems to be implanted and regenerate bone or in stem cells therapy.Copyright © 2023 Oliver-Cervelló, Martin-Gómez, Gonzalez-Garcia, Salmeron-Sanchez, Ginebra and Mas-Moruno.

JTD Keywords: biomaterials, cross-linking, dwiva, functionalization, hydrogel, integrin, kinetics, marrow stromal cells, matrices, multifunctionality, myogenic differentiation, osteogenic differentiation, regeneration, stem-cells, Biomimetic peptides, Dwiva, Functionalization, Hydrogel, Multifunctionality, Osteogenic differentiation, Poly(ethylene glycol) hydrogels

Collagen VI-related dystrophies (COL6-RDs) are a group of rare congenital neuromuscular dystrophies that represent a continuum of overlapping clinical phenotypes that go from the milder Bethlem myopathy (BM) to the severe Ullrich congenital muscular dystrophy, for which there is no effective treatment. Mutations in one of the three Collagen VI genes alter the incorporation of this protein into the extracellular matrix (ECM), affecting the assembly and the structural integrity of the whole fibrillar network. Clinical hallmarks of COL6-RDs are secondary to the ECM disruption and include muscle weakness, proximal joint contractures, and distal hyperlaxity. Although some traits have been identified in patients’ ECMs, a correlation between the ECM features and the clinical phenotype has not been established, mainly due to the lack of predictive and reliable models of the pathology. Herein, we engineered a new personalized pre-clinical model of COL6-RDs using cell-derived matrices (CDMs) technology to better recapitulate the complexity of the native scenario. We found that CDMs from COL6-RD patients presented alterations in ECM structure and composition, showing a significantly decreased Collagen VI secretion, especially in the more severe phenotypes, and a decrease in Fibrillin-1 inclusion. Next, we examined the Collagen VI-mediated deposition of Fibronectin in the ECM, finding a higher alignment, length, width, and straightness than in patients with COL6-RDs. Overall, these results indicate that CDMs models are promising tools to explore the alterations that arise in the composition and fibrillar architecture due to mutations in Collagen VI genes, especially in early stages of matrix organization. Ultimately, CDMs derived from COL6-RD patients may become relevant pre-clinical models, which may help identifying novel biomarkers to be employed in the clinics and to investigate novel therapeutic targets and treatments. Copyright © 2022 Almici, Chiappini, López-Márquez, Badosa, Blázquez, Caballero, Montero, Natera-de Benito, Nascimento, Roldán, Lagunas, Jiménez-Mallebrera and Samitier.

JTD Keywords: alpha-3 chain, binding, collagen vi related muscular dystrophy, decellularisation, decellularized matrices, deficiency, expression, extracellular matrix, fibroblasts, fibronectin, in vitro model, patient-derived ecms, skeletal-muscle, ullrich, Cell-derived matrices, Collagen, Collagen vi related muscular dystrophy, Decellularisation, Decellularization, Extracellular matrices, Extracellular matrix, Genes, In vitro model, In-vitro, In-vitro models, Matrix, Matrix model, Muscular dystrophy, Pathology, Patient-derived ecm, Patient-derived ecms, Pre-clinical

Decellularization procedures have been developed and optimized for the entire organ or tissue blocks, by either perfusion of decellularizing agents through the tissue’s vasculature or submerging large sections in decellularizing solutions. However, some research aims require the analysis of native as well as decellularized tissue slices side by side, but an optimal protocol has not yet been established to address this need. Thus, the main goal of this work was to develop a fast and efficient decellularization method for tissue slices—with an emphasis on lung—while attached to a glass slide. To this end, different decellularizing agents were compared for their effectiveness in cellular removal while preserving the extracellular matrix. The intensity of DNA staining was taken as an indicator of remaining cells and compared to untreated sections. The presence of collagen, elastin and laminin were quantified using immunostaining and signal quantification. Scaffolds resulting from the optimized protocol were mechanically characterized using atomic force microscopy. Lung scaffolds were recellularized with mesenchymal stromal cells to assess their biocompatibility. Some decellularization agents (CHAPS, triton, and ammonia hydroxide) did not achieve sufficient cell removal. Sodium dodecyl sulfate (SDS) was effective in cell removal (1% remaining DNA signal), but its sharp reduction of elastin signal (only 6% remained) plus lower attachment ratio (32%) singled out sodium deoxycholate (SD) as the optimal treatment for this application (6.5% remaining DNA signal), due to its higher elastin retention (34%) and higher attachment ratio (60%). Laminin and collagen were fully preserved in all treatments. The SD decellularization protocol was also successful for porcine and murine (mice and rat) lungs as well as for other tissues such as the heart, kidney, and bladder. No significant mechanical differences were found before and after sample decellularization. The resulting acellular lung scaffolds were shown to be biocompatible (98% cell survival after 72 h of culture). This novel method to decellularize tissue slices opens up new methodological possibilities to better understand the role of the extracellular matrix in the context of several diseases as well as tissue engineering research and can be easily adapted for scarce samples like clinical biopsies. Copyright © 2022 Narciso, Ulldemolins, Júnior, Otero, Navajas, Farré, Gavara and Almendros.

JTD Keywords: biocompatibility, bioscaffold recellularization, decellularization, extracellular matrix, flow, impact, lung, scaffolds, tissue slices, Ammonia, Bio-scaffolds, Biocompatibility, Biological organs, Bioscaffold recellularization, Cell removal, Cells, Collagen, Cytology, Decellularization, Dna, Dna signals, Elastin, Extracellular matrices, Extracellular matrix, Extracellular-matrix, Glycoproteins, Laminin, Lung, Mammals, Recellularization, Scaffolds (biology), Sodium deoxycholate, Sulfur compounds, Tissue, Tissue slice, Tissue slices

The dizzying life of the homeostatic intestinal epithelium is governed by a complex interplay between fate, form, force and function. This interplay is beginning to be elucidated thanks to advances in intravital and ex vivo imaging, organoid culture, and biomechanical measurements. Recent discoveries have untangled the intricate organization of the forces that fold the monolayer into crypts and villi, compartmentalize cell types, direct cell migration, and regulate cell identity, proliferation and death. These findings revealed that the dynamic equilibrium of the healthy intestinal epithelium relies on its ability to precisely coordinate tractions and tensions in space and time. In this review, we discuss recent findings in intestinal mechanobiology, and highlight some of the many fascinating questions that remain to be addressed in this emerging field.Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.

JTD Keywords: crypt fission, designer matrices, differentiation, growth, gut, migration, model, scaffold, tissue mechanics, Biophysics, Cell migration, Cell movement, Cell proliferation, Ex vivo study, Human tissue, Intestinal mucosa, Intestine epithelium, Monolayer culture, Organoid, Organoids, Review, Stem-cell, Tension, Traction therapy

The mechanical signals sensed by the alveolar cells through the changes in the local matrix stiffness of the extracellular matrix (ECM) are determinant for regulating cellular functions. Therefore, the study of the mechanical response of lung tissue becomes a fundamental aspect in order to further understand the mechanosensing signals perceived by the cells in the alveoli. This study is focused on the development of a finite element (FE) model of a decellularized rat lung tissue strip, which reproduces accurately the mechanical behaviour observed in the experiments by means of a tensile test. For simulating the complex structure of the lung parenchyma, which consists of a heterogeneous and non-uniform network of thin-walled alveoli, a 3D model based on a Voronoi tessellation is developed. This Voronoi-based model is considered very suitable for recreating the geometry of cellular materials with randomly distributed polygons like in the lung tissue. The material model used in the mechanical simulations of the lung tissue was characterized experimentally by means of AFM tests in order to evaluate the lung tissue stiffness on the micro scale. Thus, in this study, the micro (AFM test) and the macro scale (tensile test) mechanical behaviour are linked through the mechanical simulation with the 3D FE model based on Voronoi tessellation. Finally, a micro-mechanical FE-based model is generated from the Voronoi diagram for studying the stiffness sensed by the alveolar cells in function of two independent factors: the stretch level of the lung tissue and the geometrical position of the cells on the extracellular matrix (ECM), distinguishing between pneumocyte type I and type II. We conclude that the position of the cells within the alveolus has a great influence on the local stiffness perceived by the cells. Alveolar cells located at the corners of the alveolus, mainly type II pneumocytes, perceive a much higher stiffness than those located in the flat areas of the alveoli, which correspond to type I pneumocytes. However, the high stiffness, due to the macroscopic lung tissue stretch, affects both cells in a very similar form, thus no significant differences between them have been observed. © 2021 The Authors

JTD Keywords: rat, scaffolds, stiffness, Afm, Animal cell, Animal experiment, Animal model, Animal tissue, Article, Biological organs, Cell function, Cells, Computational geometry, Cytology, Extracellular matrices, Extracellular matrix, Extracellular-matrix, Geometry, High stiffness, Human, Lung alveolus cell type 1, Lung alveolus cell type 2, Lung parenchyma, Lung tissue, Male, Mechanical behavior, Mechanical modeling, Mechanical simulations, Mechanosensing, Model-based opc, Nonhuman, Physical model, Rat, Rigidity, Stiffness, Stiffness matrix, Tensile testing, Thin walled structures, Three dimensional finite element analysis, Tissue, Type ii, Voronoi tessellations

Most morphogenetic and pathological processes are driven by cells responding to the surrounding matrix, such as its composition, architecture, and mechanical properties. Despite increasing evidence for the role of extracellular matrix (ECM) in tissue and disease development, many in vitro substitutes still fail to effectively mimic the native microenvironment. We established a novel method to produce macroscale (>1 cm) mesenchymal cell-derived matrices (CDMs) aimed to mimic the fibrotic tumor microenvironment surrounding epithelial cancer cells. CDMs are produced by human adipose mesenchymal stem cells cultured in sacrificial 3D scaffold templates of fibronectin-coated poly-lactic acid microcarriers (MCs) in the presence of macromolecular crowders. We showed that decellularized CDMs closely mimic the fibrillar protein composition, architecture, and mechanical properties of human fibrotic ECM from cancer masses. CDMs had highly reproducible composition made of collagen types I and III and fibronectin ECM with tunable mechanical properties. Moreover, decellularized and MC-free CDMs were successfully repopulated with cancer cells throughout their 3D structure, and following chemotherapeutic treatment, cancer cells showed greater doxorubicin resistance compared to 3D culture in collagen hydrogels. Collectively, these results support the use of CDMs as a reproducible and tunable tool for developing 3D in vitro cancer models.

JTD Keywords: 3d cell-derived matrices, adipose mesenchymal stem cells, collagen matrix, colorectal adenocarcinoma, cytotoxicity assay, deposition, expansion, extracellular microenvironment, extracellular-matrix, fibronectin, growth, macromolecular crowders, microcarriers, scaffolds, tissue, 3d cell-derived matrices, Adipose mesenchymal stem cells, Cytotoxicity assay, Extracellular microenvironment, Macromolecular crowders, Mesenchymal stem-cells, Microcarriers

Regenerative medicine and disease models have evolved in recent years from two to three dimensions, providing in vitro constructs that are more similar to in vivo tissues. By mimicking native tissues, cell-derived matrices (CDMs) have emerged as new modifiable extracellular matrices for a variety of tissues, allowing researchers to study basic cellular processes in tissue-like structures, test tissue regeneration approaches, and model disease development. In this review, different fabrication techniques and characterization methods of CDMs are presented and examples of their application in cell behavior studies, tissue regeneration, and disease models are provided. In addition, future guidelines and perspectives in the field of CDMs are discussed.

JTD Keywords: 3D models, Biomaterials, Cell-derived matrices, Extracellular matrix, Personalized therapies

Neuroblastoma (NB) is a malignant tumor that affects the peripheral nervous system and represents one of the most frequent cancers in infants. Its prognosis is poor in older patients and the presence of genetic abnormalities. Metastasis is often present at the time of diagnosis, making treatment more intensive and unsuccessful. Poor prognosis and variable treatment efficacy require a better understanding of the underlying biology. Evidence has shown that the tumor microenvironment is the characteristic of tumor malignancy and progression. A more highly differentiated tissue phenotype represents a positive prognostic marker, while the tumoral tissue is characterized by a distinct composition and morphology of the extracellular matrix (ECM). In this chapter, we discuss the application of decellularized cell-derived matrices (CDMs) to model in vitro the morphology of the ECM encountered in histological hallmarks of NB patients. This technique allows for the in vitro reproduction of the fine structure and composition of native microenvironments. Because of recent advances in culture systems and decellularization techniques, it is possible to engineer CDM composition and microarchitecture to produce differentiated models of tissue niches. The final goal is to repopulate the “scaffold” with malignant NB cells for drug screening and target discovery applications, studying the impact of patient-inspired tissues on signaling, migration, and tissue remodeling.

JTD Keywords: Neuroblastoma, Cancer, Bioengineering, Tumor microenvironment, Cell-derived matrices, Decellularization

Directed cell migration is essential for many biological processes, such as embryonic development or cancer progression. Cell contractility and adhesion to the extracellular matrix are known to regulate cell locomotion machinery. However, the cross-talk between extrinsic and intrinsic factors at the molecular level on the biophysical mechanism of three dimensional (3D)-directed cell migration is still unclear. In this work, a novel physiologically relevant in vitro model of the extracellular microenvironment is used to reveal how the topological anisotropy of the extracellular matrix synergizes with actomyosin contractility to modulate directional cell migration morphodynamics. This study shows that cells seeded on polarized 3D matrices display asymmetric protrusion morphodynamics and in-vivo-like phenotypes. It is found that matrix anisotropy significantly enhances cell directionality, but strikingly, not the invasion distance of cells. In Rho-inhibited cells, matrix anisotropy counteracts the lack of actomyosin-driven forces to stabilize cell directionality suggesting a myosin-II-independent mechanism for cell guidance. Finally, this study shows that on isotropic 3D environments, cell directionality is independent of actomyosin contractility. Altogether, this study provides novel quantitative data on the biomechanical regulation of directional cell motion and shows the important regulatory role of matrix anisotropy and actomyosin forces to guide cell migration in 3D microenvironments.

JTD Keywords: Anisotropy, Directed cell migration, Extracellular matrices, Migration modes, Three dimensional microenvironments

The interaction of cells with their natural environment influences a large variety of cellular phenomena, including cell adhesion, proliferation, and migration. The complex extracellular matrix network has challenged the attempts to replicate in vitro the heterogeneity of the cell environment and has threatened, in general, the relevance of in vitro studies. In this work, we describe a new and extremely versatile approach to generate native-like extracellular matrices with controlled morphologies for the in vitro study of cellular processes. This general approach combines the confluent culture of fibroblasts with microfabricated guiding templates to direct the three-dimensional growth of well-defined extracellular networks which recapitulate the structural and biomolecular complexity of features typically found in vivo. To evaluate its performance, we studied fundamental cellular processes, including cell cytoskeleton organization, cell-matrix adhesion, proliferation, and protrusions morphodynamics. In all cases, we found striking differences depending on matrix architecture and, in particular, when compared to standard two-dimensional environments. We also assessed whether the engineered matrix networks influenced cell migration dynamics and locomotion strategy, finding enhanced migration efficiency for cells seeded on aligned matrices. Altogether, our methodology paves the way to the development of high-performance models of the extracellular matrix for potential applications in tissue engineering, diagnosis, or stem-cell biology.

JTD Keywords: Biomimetics, Cell migration, Engineered cell-derived matrices, Extracellular matrix, In vitro model

Changes in the left ventricle function produce alternans in the hemodynamic and electric behavior of the cardiovascular system. A total of 49 cardiomyopathy patients have been studied based on the blood pressure signal (BP), and were classified according to the left ventricular ejection fraction (LVEF) in low risk (LR: LVEF>35%, 17 patients) and high risk (HR: LVEF≤35, 32 patients) groups. We propose to characterize these patients using a linear and a nonlinear methods, based on the spectral estimation and the recurrence plot, respectively. From BP signal, we extracted each systolic time interval (STI), upward systolic slope (BPsl), and the difference between systolic and diastolic BP, defined as pulse pressure (PP). After, the best subset of parameters were obtained through the sequential feature selection (SFS) method. According to the results, the best classification was obtained using a combination of linear and nonlinear features from STI and PP parameters. For STI, the best combination was obtained considering the frequency peak and the diagonal structures of RP, with an area under the curve (AUC) of 79%. The same results were obtained when comparing PP values. Consequently, the use of combined linear and nonlinear parameters could improve the risk stratification of cardiomyopathy patients.

JTD Keywords: Feature extraction, Blood pressure, Heart rate, Estimation, Data mining, Covariance matrices, Hospitals

In bone regeneration, silicon-based calcium phosphate glasses (Bioglasses) have been widely used since the 1970s. However, they dissolve very slowly because of their high amount of Si (SiO2 > 45%). Recently, our group has found that calcium ions released by the degradation of glasses in which the job of silicon is done by just 5% of TiO2 are effective angiogenic promoters, because of their stimulation of a cell-membrane calcium sensing receptor (CaSR). Based on this, other focused tests on angiogenesis have found that Bioglasses also have the potential to be angiogenic promoters even with high contents of silicon (80%); however, their slow degradation is still a problem, as the levels of silicon cannot be decreased any lower than 45%. In this work, we propose a new generation of hybrid organically modified glasses, ormoglasses, that enable the levels of silicon to be reduced, therefore speeding up the degradation process. Using electrospinning as a faithful way to mimic the extracellular matrix (ECM), we successfully produced hybrid fibrous mats with three different contents of Si (40, 52, and 70%), and thus three different calcium ion release rates, using an ormoglass–polycaprolactone blend approach. These mats offered a good platform to evaluate different calcium release rates as osteogenic promoters in an in vivo subcutaneous environment. Complementary data were collected to complement Ca2+ release analysis, such as stiffness evaluation by AFM, ζ-potential, morphology evaluation by FESEM, proliferation and differentiation analysis, as well as in vivo subcutaneous implantations. Material and biological characterization suggested that compositions of organic/inorganic hybrid materials with a Si content equivalent to 40%, which were also those that released more calcium, were osteogenic. They also showed a greater ability to form blood vessels. These results suggest that Si-based ormoglasses can be considered an efficient tool for calcium release modulation, which could play a key role in the angiogenic promoting process.

JTD Keywords: Biological materials, Blood vessels, Calcium, Electrospinning, Glass, Hybrid materials, Silicon oxides, Sol-gel process, Sol-gels, Angiogenesis, Biological characterization, Calcium phosphate glass, Calcium-sensing receptors, Degradation process, Extracellular matrices, Organic/inorganic hybrid materials, ormoglasses, Silicon

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.

JTD Keywords: Ageing, Atomic force microscopy, Decellularization, Lung fibrosis, Tissue engineering, Atomic force microscopy, Biological organs, Peptides, Sodium dodecyl sulfate, Sodium sulfate, Tissue engineering, Ageing, Decellularization, Extracellular matrices, Healthy controls, Inhomogeneities, Lung fibrosis, Micro-mechanical, Statistically significant difference, Mammals, bleomycin, adventitia, animal experiment, animal model, article, atomic force microscopy, bleomycin-induced pulmonary fibrosis, cell fate, controlled study, extracellular matrix, female, intima, lung alveolus, lung fibrosis, lung mechanics, mechanical probe, microenvironment, mouse, nonhuman, pleura, priority journal, rigidity, tissue engineering