Actin-templated Structures: Nature's Way to Hierarchical Surface Patterns (Gecko's Setae as Case Study).

The hierarchical design of the toe pad surface in geckos and its reversible adhesiveness have inspired material scientists for many years. Micro- and nano-patterned surfaces with impressive adhesive performance have been developed to mimic gecko's properties. While the adhesive performance achieved in some examples has surpassed living counterparts, the durability of the fabricated surfaces is limited and the capability to self-renew and restore function-inherent to biological systems-is unimaginable. Here the morphogenesis of gecko setae using skin samples from the Bibron´s gecko (Chondrodactylus bibronii) is studied. Gecko setae develop as specialized apical differentiation structures at a distinct cell-cell layer interface within the skin epidermis. A primary role for F-actin and microtubules as templating structural elements is necessary for the development of setae's hierarchical morphology, and a stabilization role of keratins and corneus beta proteins is identified. Setae grow from single cells in a bottom layer protruding into four neighboring cells in the upper layer. The resulting multicellular junction can play a role during shedding by facilitating fracture of the cell-cell interface and release of the high aspect ratio setae. The results contribute to the understanding of setae regeneration and may inspire future concepts to bioengineer self-renewable patterned adhesive surfaces.

In Vitro Entero-Capillary Barrier Exhibits Altered Inflammatory and Exosomal Communication Pattern after Exposure to Silica Nanoparticles.

The intestinal microvasculature (iMV) plays multiple pathogenic roles during chronic inflammatory bowel disease (IBD). The iMV acts as a second line of defense and is, among other factors, crucial for the innate immunity in the gut. It is also the therapeutic location in IBD targeting aggravated leukocyte adhesion processes involving ICAM-1 and E-selectin. Specific targeting is stressed via nanoparticulate drug vehicles. Evaluating the iMV in enterocyte barrier models in vitro could shed light on inflammation and barrier-integrity processes during IBD. Therefore, we generated a barrier model by combining the enterocyte cell line Caco-2 with the microvascular endothelial cell line ISO-HAS-1 on opposite sides of a transwell filter-membrane under culture conditions which mimicked the physiological and inflamed conditions of IBD. The IBD model achieved a significant barrier-disruption, demonstrated via transepithelial-electrical resistance (TER), permeability-coefficient (Papp) and increase of sICAM sE-selectin and IL-8. In addition, the impact of a prospective model drug-vehicle (silica nanoparticles, aSNP) on ongoing inflammation was examined. A decrease of sICAM/sE-selectin was observed after aSNP-exposure to the inflamed endothelium. These findings correlated with a decreased secretion of ICAM/E-selectin bearing exosomes/microvesicles, as evaluated via ELISA. Our findings indicate that aSNP treatment of the inflamed endothelium during IBD may hamper exosomal/microvesicular systemic communication.

A responsive human triple-culture model of the air-blood barrier: incorporation of different macrophage phenotypes.

Current pulmonary research underlines the relevance of the alveolar macrophage (AM) integrated in multicellular co-culture-systems of the respiratory tract to unravel, for example, the mechanisms of tissue regeneration. AMs demonstrate a specific functionality, as they inhabit a unique microenvironment with high oxygen levels and exposure to external hazards. Healthy AMs display an anti-inflammatory phenotype, prevent hypersensitivity to normally innocuous contaminants and maintain tissue homeostasis in the alveolus. To mirror the actual physiological function of the AM, we developed three different polarized [classically activated (M1) and alternatively activated (M2wh , wound-healing; M2reg , regulatory)] macrophage models using a mixture of differentiation mediators, as described in the current literature. To test their immunological impact, these distinct macrophage phenotypes were seeded on to the epithelial layer of an established in vitro air-blood barrier co-culture, consisting of alveolar epithelial cells A549 or H441 and microvascular endothelial cells ISO-HAS-1 on the opposite side of a Transwell filter-membrane. IL-8 and sICAM release were measured as functionality parameters after LPS challenge. The M1 model itself already provoked a severe inflammatory-like response of the air-blood barrier co-culture, thus demonstrating its potential as a useful in vitro model for inflammatory lung diseases. The two M2 models represent a 'non-inflammatory' phenotype but still showed the ability to trigger inflammation following LPS challenge. Hence, the latter could be used to establish a quiescent, physiological in vitro air-blood model. Thus, the more complex differentiation protocol developed in the present study provides a responsive in vitro triple-culture model of the air-blood-barrier that mimics AM features as they occur in vivo. © 2015 The Authors Journal of Tissue Engineering and Regenerative Medicine Published by John Wiley & Sons, Ltd.

The role of the intestinal microvasculature in inflammatory bowel disease: studies with a modified Caco-2 model including endothelial cells resembling the intestinal barrier in vitro.

The microvascular endothelium of the gut barrier plays a crucial role during inflammation in inflammatory bowel disease. We have modified a commonly used intestinal cell model based on the Caco-2 cells by adding microvascular endothelial cells (ISO-HAS-1). Transwell filters were used with intestinal barrier-forming Caco-2 cells on top and the ISO-HAS-1 on the bottom of the filter. The goal was to determine whether this coculture mimics the in vivo situation more closely, and whether the model is suitable to evaluate interactions of, for example, prospective nanosized drug vehicles or contrast agents with this coculture in a physiological and inflamed state as it would occur in inflammatory bowel disease. We monitored the inflammatory responsiveness of the cells (release of IL-8, soluble intercellular adhesion molecule 1, and soluble E-selectin) after exposure to inflammatory stimuli (lipopolysaccharide, TNF-α, INF-γ, IL1-ß) and a nanoparticle (Ba/Gd: coprecipitated BaSO4 and Gd(OH)3), generally used as contrast agents. The barrier integrity of the coculture was evaluated via the determination of transepithelial electrical resistance and the apparent permeability coefficient (Papp) of NaFITC. The behavior of the coculture Caco-1/ISO-HAS-1 was compared to the respective monocultures Caco-2 and ISO-HAS-1. Based on transepithelial electrical resistance, the epithelial barrier integrity of the coculture remained stable during incubation with all stimuli, whereas the Papp decreased after exposure to the cytokine mixture (TNF-α, INF-γ, IL1-ß, and Ba/Gd). Both the endothelial and epithelial monocultures showed a high inflammatory response in both the upper and lower transwell-compartments. However, in the coculture, inflammatory mediators were only detected on the epithelial side and not on the endothelial side. Thus in the coculture, based on the Papp, the epithelial barrier appears to prevent a potential inflammatory overreaction in the underlying endothelial cells. In summary, this coculture model exhibits in vivo-like features, which cannot be observed in conventional monocultures, making the former more suitable to study interactions with external stimuli.

Development of a Bronchial Wall Model: Triple Culture on a Decellularized Porcine Trachea.

In vitro coculture models mimicking the bronchial barrier are a significant step forward in investigating the behavior and function of the upper respiratory tract mucosa. To date, mostly synthetic materials have been used as substrates to culture the cells. However, decellularized tissues provide a more in vivo-like environment based on the native extracellular matrix. In this study, an in vitro, bronchial wall coculture model has been established using a decellularized, porcine luminal trachea membrane and employing three relevant human cell types. The tissue was decellularized and placed in plastic transwell supports. The human bronchial epithelial cell line, 16HBE14o-, was seeded on the apical side of the membrane with the human lung fibroblast cell line, Wi-38, and/or the microvascular endothelial cell line, ISO-HAS-1, seeded on the basolateral side. Transepithelial electrical resistance (TER) was measured over 10 days and tight/adherens junctions (ZO-1, occludin/ß-catenin) were studied through immunofluorescence. Scanning electron microscopy (SEM) was performed to evaluate microvilli and cilia formation. All cultures grew successfully on the membrane. TER values of 555 Ω·cm(2) (±21, SEM) were achieved in the monoculture. Cocultures with fibroblasts reached 565 Ω·cm(2) (±41, SEM), with endothelial cells at 638 Ω·cm(2) (±37, SEM), and the triple culture achieved 552 Ω·cm(2) (±38, SEM). ZO-1, occludin, and ß-catenin were expressed in 16HBE14o- under all culture conditions. Using SEM, a dense microvilli population was found. Prominent cell-cell contacts and clusters of emerging cilia could be identified. Fibroblasts and endothelial cells strengthened the formation of a tight barrier by the 16HBE14o-. Thus, the coculture of three relevant cell types in combination with native decellularized scaffolds as a substrate approaches more closely the in vivo situation and could be used to study mechanisms of upper respiratory damage and regeneration.

Pulmonary surfactant augments cytotoxicity of silica nanoparticles: Studies on an in vitro air-blood barrier model.

The air-blood barrier is a very thin membrane of about 2.2 µm thickness and therefore represents an ideal portal of entry for nanoparticles to be used therapeutically in a regenerative medicine strategy. Until now, numerous studies using cellular airway models have been conducted in vitro in order to investigate the potential hazard of NPs. However, in most in vitro studies a crucial alveolar component has been neglected. Before aspirated NPs encounter the cellular air-blood barrier, they impinge on the alveolar surfactant layer (10-20 nm in thickness) that lines the entire alveolar surface. Thus, a prior interaction of NPs with pulmonary surfactant components will occur. In the present study we explored the impact of pulmonary surfactant on the cytotoxic potential of amorphous silica nanoparticles (aSNPs) using in vitro mono- and complex coculture models of the air-blood barrier. Furthermore, different surface functionalisations (plain-unmodified, amino, carboxylate) of the aSNPs were compared in order to study the impact of chemical surface properties on aSNP cytotoxicity in combination with lung surfactant. The alveolar epithelial cell line A549 was used in mono- and in coculture with the microvascular cell line ISO-HAS-1 in the form of different cytotoxicity assays (viability, membrane integrity, inflammatory responses such as IL-8 release). At a distinct concentration (100 µg/mL) aSNP-plain displayed the highest cytotoxicity and IL-8 release in monocultures of A549. aSNP-NH2 caused a slight toxic effect, whereas aSNP-COOH did not exhibit any cytotoxicity. In combination with lung surfactant, aSNP-plain revealed an increased cytotoxicity in monocultures of A549, aSNP-NH2 caused a slightly augmented toxic effect, whereas aSNP-COOH did not show any toxic alterations. A549 in coculture did not show any decreased toxicity (membrane integrity) for aSNP-plain in combination with lung surfactant. However, a significant augmented IL-8 release was observed, but no alterations in combination with lung surfactant. The augmented aSNP toxicity with surfactant in monocultures appears to depend on the chemical surface properties of the aSNPs. Reactive silanol groups seem to play a crucial role for an augmented toxicity of aSNPs. The A549 cells in the coculture seem to be more robust towards aSNPs, which might be a result of a higher differentiation and polarization state due the longer culture period.