Rationale: Extracellular matrix (ECM) is a dynamic tissue that contributes to organ integrity and function, and its regulation of cell phenotype is a major aspect of cell biology. However, standard in vitro culture approaches are of unclear physiologic relevance because they do not mimic the compositional, architectural, or distensible nature of a living organ. In the lung, fibroblasts exist in ECM-rich interstitial spaces and are key effectors of lung fibrogenesis. Objectives: To better address how ECM influences fibroblast phenotype in a disease-specific manner, we developed a culture system using acellular human normal and fibrotic lungs. Methods: Decellularization was achieved using treatment with detergents, salts, and DNase. The resultant matrices can be sectioned as uniform slices within which cells were cultured. Measurements and Main Results: We report that the decellularization process effectively removes cellular and nuclear material while retaining native dimensionality and stiffness of lung tissue. We demonstrate that lung fibroblasts reseeded into acellular lung matrices can be subsequently assayed using conventional protocols; in this manner we show that fibrotic matrices clearly promote transforming growth factor-b-independent myofibroblast differentiation compared with normal matrices. Furthermore, comprehensive analysis of acellular matrix ECM details significant compositional differences between normal and fibrotic lungs, paving the way for further study of novel hypotheses. Conclusions: This methodology is expected to allow investigation of important ECM-based hypotheses in human tissues and permits future scientific exploration in an organ-and disease-specific manner.

Engineered polyethylene glycol-maleimide matrices for regenerative medicine exhibit improved reaction efficiency and wider range of Young’s moduli by utilizing maleimide cross-linking chemistry. This hydrogel chemistry is advantageous for cell delivery due to the mild reaction that occurs rapidly enough for in situ delivery, while easily lending itself to “plug-and-play” design variations such as incorporation of enzyme-cleavable cross-links and cell-adhesion peptides.

The mechanical properties of the extracellular matrix have recently been shown to promote myofibroblast differentiation and lung fibrosis. Mechanisms by which matrix stiffness regulates myofibroblast differentiation are not fully understood. The goal of this study was to determine the intrinsic mechanisms of mechanotransduction in the regulation of matrix stiffness-induced myofibroblast differentiation. A well established polyacrylamide gel system with tunable substrate stiffness was used in this study. Megakaryoblastic leukemia factor-1 (MKL1) nuclear translocation was imaged by confocal immunofluorescent microscopy. The binding of MKL1 to the a-smooth muscle actin (a-SMA) gene promoter was quantified by quantitative chromatin immunoprecipitation assay. Normal human lung fibroblasts responded to matrix stiffening with changes in actin dynamics that favor filamentous actin polymerization. Actin polymerization resulted in nuclear translocation of MKL1, a serum response factor coactivator that plays a central role in regulating the expression of fibrotic genes, including a-SMA, a marker for myofibroblast differentiation. Mouse lung fibroblasts deficient in Mkl1 did not respond to matrix stiffening with increased a-SMA expression, whereas ectopic expression of human MKL1 cDNA restored the ability of Mkl1 null lung fibroblasts to express a-SMA. Furthermore, matrix stiffening promoted production and activation of the small GTPase RhoA, increased Rho kinase (ROCK) activity, and enhanced fibroblast contractility. Inhibition of RhoA/ROCK abrogated stiff matrix-induced actin cytoskeletal reorganization, MKL1 nuclear translocation, and myofibroblast differentiation. This study indicates that actin cytoskeletal remodeling-mediated activation of MKL1 transduces mechanical stimuli from the extracellular matrix to a fibrogenic program that promotes myofibroblast differentiation, suggesting an intrinsic mechanotransduction mechanism.Keywords: lung fibrosis; transcription factor; a-smooth muscle actinMyofibroblasts are a key effector cell type that manifests connective tissue remodeling after lung injury (1, 2). These cells are responsible for excessive extracellular matrix (ECM) deposition in idiopathic pulmonary fibrosis (IPF). Fibroblasts and mesenchymal cells are a major cellular source of myofibroblasts (2). Acquisition of a smooth muscle actin (a-SMA) expression characterizes fibroblast-tomyofibroblast differentiation. Recent studies suggest that matrix stiffness, a measure of matrix resistance to mechanical deformation, regulates myofibroblast differentiation (3). Stiff matrix-induced myofibroblast differentiation has been extensively reported in fibroblasts isolated from heart (4), aortic valves (5), lung (6-8), liver (9, 10), and gingiva (11). Despite this, the molecular mechanisms by which matrix stiffness regulates myofibroblast differentiation are not well understood. A previous study demonstrates that myofibroblast contractioninduced matrix latent TGF-b1 activation requires stiffened matrix (7). Because activated T...

The extracellular matrix (ECM) exerts powerful control over many cellular phenomena, including stem cell differentiation. As such, design and modulation of ECM analogs to ligate specific integrin is a promising approach to control cellular processes in vitro and in vivo for regenerative medicine strategies. Although fibronectin (FN), a crucial ECM protein in tissue development and repair, and its RGD peptide are widely used for cell adhesion, the promiscuity with which they engage integrins leads to difficulty in control of receptor-specific interactions. Recent simulations of force-mediated unfolding of FN domains and sequences analysis of human versus mouse FN suggest that the structural stability of the FN's central cell-binding domains (FN III9-10) affects its integrin specificity. Through production of FN III9-10 variants with variable stabilities, we obtained ligands that present different specificities for the integrin α 5 β 1 and that can be covalently linked into fibrin matrices. Here, we demonstrate the capacity of α 5 β 1 integrin-specific engagement to influence human mesenchymal stem cell (MSC) behavior in 2D and 3D environments. Our data indicate that α 5 β 1 has an important role in the control of MSC osteogenic differentiation. FN fragments with increased specificity for α 5 β 1 versus α v β 3 results in significantly enhanced osteogenic differentiation of MSCs in 2D and in a clinically relevant 3D fibrin matrix system, although attachment/spreading and proliferation were comparable with that on full-length FN. This work shows how integrin-dependant cellular interactions with the ECM can be engineered to control stem cell fate, within a system appropriate for both 3D cell culture and tissue engineering.

Fibroblasts consist of heterogeneous subpopulations that have distinct roles in fibrotic responses. Previously we reported enhanced proliferation in response to fibrogenic growth factors and selective activation of latent transforming growth factor (TGF)-␤ in fibroblasts lacking cell surface expression of Thy-1 glycoprotein, suggesting that Thy-1 modulates the fibrogenic potential of fibroblasts. Here we report that compared to controls Thy-1؊/؊ C57BL/6 mice displayed more severe histopathological lung fibrosis, greater accumulation of lung collagen, and increased TGF-␤ activation in the lungs 14 days after intratracheal bleomycin. The majority of cells demonstrating TGF-␤ activation and myofibroblast differentiation in bleomycin-induced lesions were Thy-1-negative. Histological sections from patients with idiopathic pulmonary fibrosis demonstrated absent Thy-1 staining within fibroblastic foci. Normal lung fibroblasts, in both mice and humans, were predominantly Thy-1-positive. The fibrogenic cytokines interleukin-1 and tumor necrosis factor-␣ induced loss of fibroblast Thy-1 surface expression in vitro, which was associated with Thy-1 shedding, Smad phosphorylation, and myofibroblast differentiation. These results suggest that fibrogenic injury promotes loss of lung fibroblast Thy-1 expression, resulting in enhanced fibrogenesis. (Am J Pathol 2005, 167:365-379) Idiopathic pulmonary fibrosis (IPF), with its histopathological signature of usual interstitial pneumonia (UIP), is a paradigmatic, but as yet primarily enigmatic example of uncontrolled fibroproliferation. IPF is remarkable for its insidious onset, dramatic histopathological and pathophysiological derangements, and relentless progression to death regardless of treatment. The etiology of IPF, and the factors that direct its dismal outcome, remain the subject of intense investigation.1 Fibroblasts are the cellular sine qua non of fibrosis in most tissues, and the histopathology of IPF underscores this observation. The histopathological feature most clearly correlated with outcome is the presence in lung of fibroblastic foci of young connective tissue, the presence of which portends death within months.2 Fibroblastic foci seem to represent the fibroproliferative leading edge of the heterogeneous areas of scarring in IPF.3-5 The myofibroblasts within these foci are clearly dysregulated in their proliferative and matrix-productive function, yet the origin of these cells, and the factors that lead to their accumulation and persistence, are unknown.Fibroblasts in most tissues are heterogeneous with respect to size, secretory profile, and surface markers. Fibroblasts within a fibrogenic milieu clearly differ from those in normal tissues. In particular, fibroblasts isolated from lungs with active fibrotic disease have increased proliferative capacity, are capable of anchorage-independent growth, and are morphologically distinct. 6 -8 Furthermore, differences among subsets of normal fibroblasts have been identified on the basis of surface markers, cytoskeleta...

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Biomaterial‐mediated inflammation and fibrosis remain a prominent challenge in designing materials to support tissue repair and regeneration. Despite the many biomaterial technologies that have been designed to evade or suppress inflammation (i.e., delivery of anti‐inflammatory drugs, hydrophobic coatings, etc.), many materials are still subject to a foreign body response, resulting in encapsulation of dense, scar‐like extracellular matrix. The primary cells involved in biomaterial‐mediated fibrosis are macrophages, which modulate inflammation, and fibroblasts, which primarily lay down new extracellular matrix. While macrophages and fibroblasts are implicated in driving biomaterial‐mediated fibrosis, the signaling pathways and spatiotemporal crosstalk between these cell types remain loosely defined. In this review, the role of M1 and M2 macrophages (and soluble cues) involved in the fibrous encapsulation of biomaterials in vivo is investigated, with additional focus on fibroblast and macrophage crosstalk in vitro along with in vitro models to study the foreign body response. Lastly, several strategies that have been used to specifically modulate macrophage and fibroblast behavior in vitro and in vivo to control biomaterial‐mediated fibrosis are highlighted.

SPARC, a 32-kDa matricellular glycoprotein, mediates interactions between cells and their extracellular matrix, and targeted deletion of Sparc results in compromised extracellular matrix in mice. Fibronectin matrix provides provisional tissue scaffolding during development and wound healing and is essential for the stabilization of mature extracellular matrix. Herein, we report that SPARC expression does not significantly affect fibronectin-induced cell spreading but enhances fibronectin-induced stress fiber formation and cell-mediated partial unfolding of fibronectin molecules, an essential process in fibronectin matrix assembly. By phage display, we identify integrin-linked kinase as a potential binding partner of SPARC and verify the interaction by co-immunoprecipitation and colocalization in vitro. Cells lacking SPARC exhibit diminished fibronectin-induced integrin-linked kinase activation and integrin-linked kinase-dependent cell-contractile signaling. Furthermore, induced expression of SPARC in SPARC-null fibroblasts restores fibronectin-induced integrin-linked kinase activation, downstream signaling, and fibronectin unfolding. These data further confirm the function of SPARC in extracellular matrix organization and identify a novel mechanism by which SPARC regulates extracellular matrix assembly.Matricellular proteins such as SPARC function as modulators of cellextracellular matrix (ECM) 2 interactions (1, 2). SPARC is considered "antiadhesive," because it does not directly support cell attachment. Moreover, it induces focal adhesion disassembly and cell rounding when the purified protein is added to spread cells (3-5). The induction of an intermediate state of cell adhesion by SPARC (6) implies a role for SPARC in the organization of ECM. Consistent with data acquired in vitro, mice with a targeted disruption of Sparc have marked developmental abnormalities in the dermis, eye, and adipose tissue (7-9) and show accelerated closure of dermal wounds (10 -11), diminished foreign body response (12), and enhanced tumor growth (13). These aberrations have been explained, in part, by altered ECM production and assembly. Specifically, tissues from SPARC-null (SϪ/Ϫ) mice contain less collagen than those from wild-type (WT) mice, and the collagen present is less mature (7). However, the mechanism by which SPARC directs ECM assembly has not been identified.The development of mature ECM requires proper formation of an organized fibronectin (Fn) matrix. The importance of Fn in the morphogenesis and patterning of tissues is established, since Fn-null mice die during early gastrulation as a result of defective cell migration (14). Integrin-linked kinase (ILK), a serine/threonine kinase that binds to the intracellular domain of ␤1 integrin immediately adjacent to the plasma membrane and is activated by ␤1 integrins and growth factors, has been shown to control the intracellular signaling cascades that influence cellular contractile elements (25). ILK interacts directly with actin and ␣-actinin-binding proteins such as th...