Expansion on stiff culture substrates activates pro-fibrotic cell programs that are retained by mechanical memory. Here, we show that priming on physiologically soft silicone substrates suppresses ...fibrogenesis and desensitizes mesenchymal stem cells (MSCs) against subsequent mechanical activation in vitro and in vivo, and identify the microRNA miR-21 as a long-term memory keeper of the fibrogenic program in MSCs. During stiff priming, miR-21 levels were gradually increased by continued regulation through the acutely mechanosensitive myocardin-related transcription factor-A (MRTF-A/MLK-1) and remained high over 2 weeks after removal of the mechanical stimulus. Knocking down miR-21 once by the end of the stiff-priming period was sufficient to erase the mechanical memory and sensitize MSCs to subsequent exposure to soft substrates. Soft priming and erasing mechanical memory following cell culture expansion protects MSCs from fibrogenesis in the host wound environment and increases the chances for success of MSC therapy in tissue-repair applications.
The mechanical memory of lung myofibroblasts Balestrini, Jenna L; Chaudhry, Sidharth; Sarrazy, Vincent ...
Integrative biology (Cambridge),
04/2012, Letnik:
4, Številka:
4
Journal Article
Recenzirano
Fibroblasts differentiate into the highly synthetic and contractile myofibroblast phenotype when exposed to substrates with an elastic modulus corresponding to pathologically stiff fibrotic tissue. ...Cellular responses to changes in substrate stiffness are typically analyzed after hours or days, which does not enable the monitoring of myofibroblast persistence, a hallmark of fibrosis. To determine long-lasting effects on the fibrotic behavior of lung fibroblasts, we followed a novel approach of explanting and repeatedly passaging fibroblasts on silicone substrates with stiffness representing various states of lung health. Fibrotic activity was determined by assaying for myofibroblast proliferation, cell contractility, expression of α-smooth muscle actin, extracellular matrix and active TGFβ1. As predicted, myofibroblast activity was low on healthy soft substrates and increased with increasing substrate stiffness. However, explanting and mechanically priming lung fibroblasts for 3 weeks on pathologically stiff substrates resulted in sustained myofibroblast activity even after the cells were returned to healthy soft cultures for 2 weeks. Such primed cells retained higher fibrotic activity than cells that had been exclusively cultured on soft substrates, and were not statistically different from cells continuously passaged on stiff surfaces. Inversely, priming lung fibroblasts for 3 weeks on soft substrates partially protected from myofibroblast activation after the shift to stiff substrates. Hence, mechano-sensed information relating to physical conditions of the local cellular environment could permanently induce fibrotic behavior of lung fibroblasts. This priming effect has important implications for the progression and persistence of aggressive fibrotic diseases such as idiopathic pulmonary fibrosis.
Extracellular matrix has manifold roles in tissue mechanics, guidance of cellular behavior, developmental biology, and regenerative medicine. Over the past several decades, various pre-clinical and ...clinical studies have shown that many connective tissues may be replaced and/or regenerated using suitable extracellular matrix scaffolds. More recently, decellularization of lung tissue has shown that gentle removal of cells can leave behind a “footprint” within the matrix that may guide cellular adhesion, differentiation and homing following cellular repopulation. Fundamental issues like understanding matrix composition and micro-mechanics remain difficult to tackle, largely because of a lack of available assays and tools for systematically characterizing intact matrix from tissues and organs. This review will critically examine the role of engineered and native extracellular matrix in tissue and lung regeneration, and provide insights into directions for future research and translation.
Abstract Lung engineering is a promising technology, relying on re-seeding of either human or xenographic decellularized matrices with patient-derived pulmonary cells. Little is known about the ...species-specificity of decellularization in various models of lung regeneration, or if species dependent cell-matrix interactions exist within these systems. Therefore decellularized scaffolds were produced from rat, pig, primate and human lungs, and assessed by measuring residual DNA, mechanical properties, and key matrix proteins (collagen, elastin, glycosaminoglycans). To study intrinsic matrix biologic cues, human endothelial cells were seeded onto acellular slices and analyzed for markers of cell health and inflammation. Despite similar levels of collagen after decellularization, human and primate lungs were stiffer, contained more elastin, and retained fewer glycosaminoglycans than pig or rat lung scaffolds. Human endothelial cells seeded onto human and primate lung tissue demonstrated less expression of vascular cell adhesion molecule and activation of nuclear factor-κB compared to those seeded onto rodent or porcine tissue. Adhesion of endothelial cells was markedly enhanced on human and primate tissues. Our work suggests that species-dependent biologic cues intrinsic to lung extracellular matrix could have profound effects on attempts at lung regeneration.
A novel method enabling the engineering of a dense and appropriately oriented heparin-containing layer on decellularized aortas has been developed. Amino groups of decellularized aortas were first ...modified to azido groups using 3-azidobenzoic acid. Azide-clickable dendrons were attached onto the azido groups through “alkyne–azide” click chemistry, affording a tenfold amplification of adhesions sites. Dendron end groups were finally decorated with end-on modified heparin chains. Heparin chains were oriented like heparan sulfate groups on native endothelial cells surface. X-ray photoelectron spectroscopy, nuclear magnetic resonance imaging, mass spectrometry and Fourier transform infrared FTIR spectroscopy were used to characterize the synthesis steps, building the final heparin layered coatings. The continuity of the heparin coating was verified using fluorescent microscopy and histological analysis. The efficacy of heparin linkage was demonstrated with factor Xa anti-thrombogenic assay and platelet adhesion studies. The results suggest that oriented heparin immobilization to decellularized aortas may improve the in vivo blood compatibility of decellularized aortas and vessels.
For an airway or a blood vessel to narrow, there must be a connected path that links the smooth muscle (SM) cells with each other, and transmits forces around the organ, causing it to constrict. ...Currently, we know very little about the mechanisms that regulate force transmission pathways in a multicellular SM ensemble. Here, we used extracellular matrix (ECM) micropatterning to study force transmission in a two-cell ensemble of SM cells. Using the two-SM cell ensemble, we demonstrate (a) that ECM stiffness acts as a switch that regulates whether SM force is transmitted through the ECM or through cell-cell connections. (b) Fluorescent imaging for adherens junctions and focal adhesions show the progressive loss of cell-cell borders and the appearance of focal adhesions with the increase in ECM stiffness (confirming our mechanical measurements). (c) At the same ECM stiffness, we show that the presence of a cell-cell border substantially decreases the overall contractility of the SM cell ensemble. Our results demonstrate that connectivity among SM cells is a critical factor to consider in the development of diseases such as asthma and hypertension.
Objective
Fibrocytes are collagen‐producing leukocytes that accumulate in patients with systemic sclerosis (SSc; scleroderma)–related interstitial lung disease (ILD) via unknown mechanisms that have ...been associated with altered expression of neuroimmune proteins. The extracellular matrix (ECM) influences cellular phenotypes. However, a relationship between the lung ECM and fibrocytes in SSc has not been explored. The aim of this study was to use a novel translational platform based on decellularized human lungs to determine whether the lung ECM of patients with scleroderma controls the development of fibrocytes from peripheral blood mononuclear cells.
Methods
We performed biomechanical evaluation of decellularized scaffolds prepared from lung explants from healthy control subjects and patients with scleroderma, using tensile testing and biochemical and proteomic analysis. Cells obtained from healthy controls and patients with SSc‐related ILD were cultured on these scaffolds, and CD45+pro‐ColIα1+ cells meeting the criteria for fibrocytes were quantified. The contribution of the neuromolecule netrin‐1 to fibrosis was assessed using neutralizing antibodies in this system and by administering bleomycin via inhalation to netrin‐1+/− mice.
Results
Compared with control lung scaffolds, lung scaffolds from patients with SSc‐related ILD showed aberrant anatomy, enhanced stiffness, and abnormal ECM composition. Culture of control cells in lung scaffolds from patients with SSc‐related ILD increased production of pro‐ColIα1+ cells, which was stimulated by enhanced stiffness and abnormal ECM composition. Cells from patients with SSc‐related ILD demonstrated increased pro‐ColIα1 responsiveness to lung scaffolds from scleroderma patients but not enhanced stiffness. Enhanced detection of netrin‐1–expressing CD14low cells in patients with SSc‐related ILD was observed, and antibody‐mediated netrin‐1 neutralization attenuated detection of CD45+pro‐ColIα1+ cells in all settings. Netrin‐1+/− mice were protected against bleomycin‐induced lung fibrosis and fibrocyte accumulation.
Conclusion
Factors present in the lung matrices of patients with scleroderma regulate fibrocyte accumulation via a netrin‐1–dependent pathway. Netrin‐1 regulates bleomycin‐induced pulmonary fibrosis in mice. Netrin‐1 might be a novel therapeutic target in SSc‐related ILD.
Nanopatterning of biomaterials is rapidly emerging as a tool to engineer cell function. Bulk metallic glasses (BMGs), a class of biocompatible materials, are uniquely suited to study nanopattern–cell ...interactions as they allow for versatile fabrication of nanopatterns through thermoplastic forming. Work presented here employs nanopatterned BMG substrates to explore detection of nanopattern feature sizes by various cell types, including cells that are associated with foreign body response, pathology, and tissue repair. Fibroblasts decreased in cell area as the nanopattern feature size increased, and fibroblasts could detect nanopatterns as small as 55 nm in size. Macrophages failed to detect nanopatterns of 150 nm or smaller in size, but responded to a feature size of 200 nm, resulting in larger and more elongated cell morphology. Endothelial cells responded to nanopatterns of 100 nm or larger in size by a significant decrease in cell size and elongation. On the basis of these observations, nondimensional analysis was employed to correlate cellular morphology and substrate nanotopography. Analysis of the molecular pathways that induce cytoskeletal remodeling, in conjunction with quantifying cell traction forces with nanoscale precision using a unique FIB-SEM technique, enabled the characterization of underlying biomechanical cues. Nanopatterns altered serum protein adsorption and effective substrate stiffness, leading to changes in focal adhesion density and compromised activation of Rho-A GTPase in fibroblasts. As a consequence, cells displayed restricted cell spreading and decreased collagen production. These observations suggest that topography on the nanoscale can be designed to engineer cellular responses to biomaterials.
The creation of decellularized organs for use in regenerative medicine requires the preservation of the organ extracellular matrix (ECM) as a means to provide critical cues for differentiation and ...migration of cells that are seeded onto the organ scaffold. The purpose of this study was to assess the influence of varying pH levels on the preservation of key ECM components during the decellularization of rat lungs. Herein, we show that the pH of the 3-(3-cholamidopropyl)dimethylammonio-1-propanesulfonate (CHAPS)-based decellularization solution influences ECM retention, cell removal, and also the potential for host response upon implantation of acellular lung tissue. The preservation of ECM components, including elastin, fibronectin, and laminin, were better retained in the lower pH conditions that were tested (pH ranges tested: 8, 10, 12); glycosaminoglycans were preserved to a higher extent in the lower pH groups as well. The DNA content following decellularization of the rat lung was inversely correlated with the pH of the decellularization solution. Despite detectible levels of cyotoskeletal proteins and significant residual DNA, tissues decellularized at pH 8 demonstrated the greatest tissue architecture maintenance and the least induction of host response of all acellular conditions. These results highlight the effect of pH on the results obtained by organ decellularization and suggest that altering the pH of the solutions used for decellularization may influence the ability of cells to properly differentiate and home to appropriate locations within the scaffold, based on the preservation of key ECM components and implantation results.
Autologous cellular therapies based on modifying T cells to express chimeric antigen receptor genes have been highly successful in treating hematological cancers. Deployment of these therapies is ...limited by the complexity and costs associated with their manufacturing. Transitioning these processes from virus-based methods for gene delivery to a non-viral method, such as electroporation, has the potential to greatly reduce cost and manufacturing time while increasing safety and efficacy. Major challenges with electroporation are the negative impacts on cell health associated with exposure to high-magnitude electric fields, and that most commercial bulk electroporators are low-precision instruments designed for manually-operated, lower-throughput batch processing of cells. Negative effects on cell health can be mitigated by use of specialized electroporation medias, but this adds processing steps, and long-term exposure to these medias can reduce transfection efficiency and post-transfection viability. To enable automated, clinical-scale production of cellular therapies using electrotransfection in specialized medias, we developed a high-precision microfluidic platform that automatically and continuously transfers cells from culture media into electroporation media using acoustophoresis, and then immediately applies electric fields from integrated electrodes. This limits cell residence time in electroporation media to seconds, and enables high transfection efficiency with minimum impact on cell viability. We tested our system by transferring primary human T cells from a standard cell media to electroporation media, and then transfecting them with mRNA encoding an mCherry fluorescent protein. We achieved a media exchange efficiency of 86% and transfection efficiency of up to 60%, with less than a 5% reduction in viability.
Our novel device acoustophoretically transfers cells from culture media to electroporation media and then electroporates them using integrated electrodes.
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