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In this study, we describe composite scaffolds composed of synthetic and natural materials with physicochemical properties suitable for tissue engineering applications. Fibrous scaffolds were co-electrospun from a blend of a synthetic biodegradable polymer (poly(lactic-co-glycolic acid), PLGA, 10% solution) and two natural proteins, gelatin (denatured collagen, 8% solution) and alpha-elastin (20% solution) at ratios of 3:1:2 and 2:2:2 (v/v/v). The resulting PLGA-gelatin-elastin (PGE) fibers were homogeneous in appearance with an average diameter of 380 +/- 80 nm, which was considerably smaller than fibers made under identical conditions from the starting materials (PLGA, 780 +/- 200 nm; gelatin, 447 +/- 123 nm; elastin, 1060 +/- 170 nm). Upon hydration, PGE fibers swelled to an average fiber diameter of 963 +/- 132 nm, but did not disintegrate. Importantly, PGE scaffolds were stable in an aqueous environment without crosslinking and were more elastic than those made of pure elastin fibers. To investigate the cytocompatibility of PGE, we cultured H9c2 rat cardiac myoblasts and rat bone marrow stromal cells (BMSCs) on fibrous PGE scaffolds. We found that myoblasts grew equally as well or slightly better on the scaffolds than on tissue-culture plastic. Microscopic evaluation confirmed that myoblasts reached confluence on the scaffold surfaces while simultaneously growing into the scaffolds. Histological characterization of the PGE constructs indicated that BMSCs penetrated into the center of scaffolds and began proliferating shortly after seeding. Our results suggest that fibrous scaffolds made of PGE and similar biomimetic blends of natural and synthetic polymers may be useful for engineering soft tissues, such as heart, lung, and blood vessels.

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In this paper, we report on engineering 3-D pulmonary tissue constructs in vitro. Primary isolates of murine embryonic day 18 fetal pulmonary cells (FPC) were comprised of a mixed population of epithelial, mesenchymal, and endothelial cells as assessed by immunohistochemistry and RT-PCR of 2-D cultures. The alveolar type II (AE2) cell phenotype in 2-D and 3-D cultures was confirmed by detection of SpC gene expression and presence of the gene product prosurfactant protein C. Three-dimensional constructs of FPC were generated utilizing Matrigel hydrogel and synthetic polymer scaffolds of poly-lactic-co-glycolic acid (PLGA) and poly-L-lactic-acid (PLLA) fabricated into porous foams and nanofibrous matrices, respectively. Three-dimensional Matrigel constructs contained alveolar forming units (AFU) comprised of cells displaying AE2 cellular ultrastructure while expressing the SpC gene and gene product. The addition of tissue-specific growth factors induced formation of branching, sacculated epithelial structures reminiscent of the distal lung architecture. Importantly, 3-D culture was necessary for inducing expression of the morphogenesis-associated distal epithelial gene fibroblast growth factor receptor 2 (FGFr2). PLGA foams and PLLA nanofiber scaffolds facilitated ingrowth of FPC, as evidenced by histology. However, these matrices did not support the survival of distal lung epithelial cells, despite the presence of tissue-specific growth factors. Our results may provide the first step on the long road toward engineering distal pulmonary tissue for augmenting and/or replacing dysfunctional native lung in diseases, such as neonatal pulmonary hypoplasia.

Semipermeable cell culture membranes are commonly used in multilayered microfluidic devices to mimic the basement membrane in vivo and to create compartmentalized microenvironments for physiological cell growth and differentiation. However, existing membranes are predominantly made up of synthetic polymers, providing limited capacity to recapitulate cellular interactions with native extracellular matrices that play a crucial role in the induction of physiological phenotypes. Here we describe a new type of cell culture membranes engineered from native extracellular matrix (ECM) materials that are thin, semipermeable, optically transparent, and amenable to integration into microfluidic cell culture devices. Facile and cost-effective fabrication of these membranes was achieved by controlled sequential steps of vitrification that transformed three-dimensional (3D) ECM hydrogels into structurally stable thin films. By modulating the composition of ECM, our technique provided a means to tune key membrane properties such as optical transparency, stiffness, and porosity. For microfluidic cell culture, we constructed a multilayered microdevice consisting of two parallel chambers separated by a thin membrane insert derived from different types of ECM. This study showed that our ECM membranes supported attachment and growth of various types of cells (epithelial, endothelial, and mesenchymal cells) under perfusion culture conditions. Our data also revealed the promotive effects of the membranes on adhesion-associated intracellular signaling that mediates cell-ECM interactions. Moreover, we demonstrated the use of these membranes for constructing compartmentalized microfluidic cell culture systems to induce physiological tissue differentiation or to replicate interfaces between different tissue types. Our approach provides a robust platform to produce and engineer biologically active cell culture substrates that serve as promising alternatives to conventional synthetic membrane inserts. This strategy may contribute to developing physiologically relevant in vitro cell culture models for a wide range of applications.