The ECM is dynamic tissue providing structural support and spatial and temporal contextual cues to cells during many processes, including embryonic development, wound repair, and homeostasis. A great deal has been learned about the role of ECM in health and disease using standard cell culture techniques, although it is clear that these approaches mimic neither the compositional heterogeneity of ECM in vivo
nor the dimensionality of tissues. As a result, data gleaned using traditional approaches may not accurately reflect cell behavior that occurs in vivo
. Along the same lines, experimental animal models using transgenic approaches have helped to elucidate the role of many ECM proteins in various disease states (34
). Unfortunately, animal models do not always recapitulate human disease, raising questions about the ability to translate results from animal to human. For example, anatomic differences in the lung between humans and rodents, such as lobar distribution, branching patterns, presence of bronchial submucosal glands, and goblet cells (37
), likely substantially influence results. Indeed, these factors may account for the lack of effective therapies for patients with idiopathic pulmonary fibrosis; numerous compounds that are efficacious in rodent models of lung fibrosis are ineffective in the human disease (11
To overcome some of these limitations, we have developed a fully humanized novel model for culturing cells in vitro
in an organ- and disease-specific manner. Our approach retains the 3D spatial orientation, relative stiffness, complex ECM composition, and architecture found in the human lung; it is likely that these features will be maintained regardless of the organ tested. In fact, prior studies using decellularized heart (15
), bone (38
), and murine lung (39
) as matrices to support regeneration imply these features remain true to the native organ, although this has not been directly shown previously. Our model also provides physiologically relevant tissue stiffness and ECM composition, as compared with the standard planar rigidity of tissue culture plastic. Moreover, it allows investigators to study the roles of ECM and cellular processes in a disease-specific manner, which is a major advance over currently used techniques.
In addition to defining a new model culture system, our data also show marked differences between the normal and (IPF) disease state, which may provide insight into disease pathogenesis. For example, we observed that IPF lung ECM markedly expresses glycosaminoglycans, matrix Gla protein, and microfibrillar-associated proteins. These observations are notable in that, although heparan sulfate– and chondroitin sulfate–containing glycosaminoglycans are known to be produced by various lung cell types (40
), their presence in IPF has only been indirectly shown. Similarly, matrix Gla proteins, although identified in TGF-α–induced rodent lung fibrosis (29
), have not previously been implicated in human lung fibrosis.
Studies of tissue fibrosis in general, and IPF specifically, clearly implicate the profibrotic effects of TGF-β (41
). Thus, there has been an intense effort to delineate the role of TGF-β in IPF and other fibrosing disorders. These efforts suggest that TGF-β promotes myofibroblast differentiation (45
), the fibroblast antiapoptotic phenotype (46
), epithelial–mesenchymal transition (47
), and ECM production (48
). However, our current work suggests that myofibroblast differentiation (as measured by expression of α-SMA and cellular fibronectin) occurs at least partially independent of TGF-β in our system. Whether this finding applies to the true in vivo
condition is not known, because our data show (Table E2) that latent TGF-β–binding proteins-2 and -4 are stripped away during the decellularization process. Regardless, evidence supports the notion that myofibroblast differentiation in the absence of TGF-β signaling can be induced in fibroblasts merely by altering the stiffness of the underlying substrate (49
). Our current work is highly concordant with a seminal finding by Liu and colleagues (49
), namely that fibroblasts seeded on polyacrylamide-crosslinked hydrogels maintained a quiescent phenotype or underwent apoptosis at low stiffness levels (0.1 to ~ 3 kPa), whereas higher stiffness levels (20–50 kPa) induced an appearance of activation characterized by accumulation of cells aligned in parallel clusters (49
). This is very similar to our own findings (), in which fibroblasts cultured in stiffer IPF matrices clearly adopted an activated myofibroblast phenotype.
The pathogenesis of IPF remains incompletely understood. In part, this is due to the lack of an animal model that faithfully recapitulates the human disease (12
). However, the bleomycin model (in which a dose of intratracheal bleomycin is instilled in rodents and fibrosis is measured 21–35 d later) remains one of the most widely used to address mechanistic questions about IPF. Although this model shares some features with human fibrosis, its complete reliance on the preceding inflammatory injury makes it a less-than-ideal model to study IPF. As proof of this shortcoming, many therapeutic agents that are shown to be successful in bleomycin-induced lung fibrosis are often ineffective in patients with IPF (50
). Thus, new models that more closely resemble the human disease are necessary to further our understanding. The in vitro
model described herein provides such a tool to begin to dissect, mechanistically, the contribution of the ECM to IPF pathogenesis. Moreover, we believe this model can be adapted to other organs and diseases as a means to enhance the translatability of biomedical research.