Connexin communication compartments and wound repair in epithelial tissue

Epithelial tissues line the lumen of tracts and ducts connecting to the external environment. They are critical in forming an interface between the internal and external environment and, following assault from environmental factors and pathogens, they must rapidly repair to maintain cellular homeostasis. These tissue networks, that range from a single cell layer, such as in airway epithelium, to highly stratified and differentiated epithelial surfaces, such as the epidermis, are held together by a junctional nexus of proteins including adherens, tight and gap junctions, often forming unique and localised communication compartments activated for localised tissue repair. This review focuses on the dynamic changes that occur in connexins, the constituent proteins of the intercellular gap junction channel, during wound-healing processes and in localised inflammation, with an emphasis on the lung and skin. Current developments in targeting connexins as corrective therapies to improve wound closure and resolve localised inflammation are also discussed. Finally, we consider the emergence of the zebrafish as a concerted whole-animal model to study, visualise and track the events of wound repair and regeneration in real-time living model systems

Thermosensitive bioartificial hydrogels as smart injectable and biocompatible systems allowing post-injection chemical crosslinking

Introduction Injectable hydrogels as carriers for drugs and/or cells have gained increasing interest in the last years due to the possibility to vehicle their payload in the desired loco through mini-invasive procedures [1, 2]. In this work, a new library of bioartificial hydrogels was designed combining the chemical versatility and reproducible physicochemical properties of a synthetic polymer with the enhanced cell adhesiveness of a natural polymer. Specifically, an amphiphilic polyurethane (PEU) bearing amino groups was first synthesised and then blended with hyaluronic acid (HA) to obtain thermosensitive bioartificial formulations. The influence of HA molecular weight on polymers miscibility was investigated as well as the thermosensitivity and the injectability of the newly designed bioartificial systems. Modification of the PEU and the HA may allow post-injection chemical crosslinking enhancing the chemical stability of the hydrogel. Experimental Methods The amphiphilic PEU was synthesised in a two step procedure under nitrogen by reacting a commercial triblock copolymer (Poloxamer 407, Poly(ethylene oxide)-Poly(propylene oxide)-Poly(ethylene oxide)) with 1,6-hexamethylene diisocyanate. Then, the prepolymer was chain extended with a diol (N-Boc diethanolamine) containing protected secondary amino groups. Infrared (IR) spectroscopy and Size Exclusion Chromatography (SEC) were then performed to assess the success of the synthesis and to evaluate PEU molecular weight, respectively. Subsequently, the synthesised PEU was subjected to an acidic treatment in chloroform/trifluoroacetic acid 90/10 V/V to remove BOC-protecting groups and the exposed secondary amino groups were quantified through a colorimetric assay (Orange Sodium salt). The synthetic component (D-DHP407) was then blended with a high (HA_400kDa) and low (HA_82kDa) molecular weight HA, reaching different weight ratios. Lastly, formulations were prepared by dissolving both polymers in physiological saline solution and then characterized in terms of thermosensitivity by means of tube investing test and gelation time test at 37 °C; injectability in the sol state through needles of different diameters (G18, G21 and G22) and cytocompatibility according to ISO10993-5. Secondary amino groups in PEU and carboxyl groups in HA may be exploited to graft functional molecules for in situ post-injection crosslinking. Results and Discussion The successful PEU synthesis was proved through IR spectroscopy by the appearance of new bands ascribed to urethane bonds, while SEC analysis gave a molecular weight in the range 30000 – 35000 Da with 1.4 polydispersity index. Secondary amino groups were quantified to be 4.5x1020 groups/g of polymer by means of Orange II Sodium Salt assay. For what concerns hydrogel preparation, D-DHP407 and HA_400kDa were mixed at 98/2, 95/5, 92/8, 88/12 and 83/17 wt. ratios. All tested formulations formed compatible blends, but, due to the high molecular weight of HA_400kDa, further increase in the natural component content highly increased system viscosity affecting injectability. Hence, D-DHP407 was blended with a low molecular weight HA (HA_82kDa) obtaining compatible and injectable blends even at 50/50 wt. ratio. Subsequently, hydrogel temperature-driven gelation was tested and all considered formulations turned out to gel within few minutes at 37 °C, thus suggesting that HA introduction did not affect PEU thermosensitivity. Regarding hydrogel injectability, all blends could not be extruded through G22 needle, while they could be injected through larger needle diameters (G21 and G18). Finally, by increasing HA content, thus decreasing the synthetic component, an increase of hydrogel biocompatibility was observed. Conclusion A new platform of thermosensitive bioartificial hydrogels was developed by blending a custom-made amphiphilic polyurethane, which ensures hydrogel thermo-responsiveness, with hyaluronic acid, responsible for an improved cytocompatibility. Furthermore, the presence of exposed secondary amino groups along PEU chains and carboxylic groups in HA chains opens the possibility to graft functional moieties to both molecules for post-injection crosslinking. Such injectable system is under development as a matrix for in situ treatment of myocardial tissue, by releasing agents promoting direct reprogramming of cardiac fibroblasts into cardiomyocytes. References [1]Zhang Z., Expert. Opin. Biol. Ther., 17:1, 49-62, 2017. [2] Boffito M. et al., J. Biomed. Mater. Res. A, 103(3):1276-90, 2015. Acknowledgement The activity has been carried within the research project BIORECAR. This project has received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreement No 772168)

Immune response of polarized cystic fibrosis airway epithelial cells infected with Influenza A virus

Background: Cystic fibrosis (CF), a genetic disease caused by mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, is characterized by dysfunction of the immune response in the airway epithelium that leads to prolonged infection, colonization and exacerbated inflammation. In this study, we determined the gene expression profile of airway epithelial cells knockdown for CFTR (CFTR KD) in response to bacterial and viral challenges. Methods: In a first approach, polarized CFTR KD and their control counterpart (CFTR CTL) cells were stimulated with P. aeruginosa-derived virulence factor flagellin. Next, we developed a model of Influenza A virus (IAV) infection in CTL and CFTR KD polarized cells. mRNA was collected for transcriptome analysis. Results: Beside the expected pro-inflammatory response, Gene Set Enrichment Analysis highlighted key molecular pathways and players involved in IAV and anti-viral interferon signaling. Although IAV replication was similar in both cell types, multiplex gene expression analysis revealed changes of key immune genes dependent on time of infection that were found to be CFTR-dependent and/or IAV-dependent. Interferons are key signaling proteins/cytokines in the antibacterial and antiviral response. To evaluate their impact on the altered gene expression profile in CFTR responses to pathogens, we measured transcriptome changes after exposure to Type I-, Type II- and Type III-interferons. Conclusions: Our findings reveal target genes in understanding the defective immune response in the CF airway epithelium in the context of viral infection. Information provided in this study would be useful to understand the dysfunctional immune response of the CF airway epithelium during infection

Three-Dimensional Microfibrous Scaffold with Aligned Topography Produced via a Combination of Melt-Extrusion Additive Manufacturing and Porogen Leaching for In Vitro Skeletal Muscle Modeling

Skeletal muscle tissue (SMT) has a highly hierarchical and anisotropic morphology, featuring aligned and parallel structures at multiple levels. Various factors, including trauma and disease conditions, can compromise the functionality of skeletal muscle. The in vitro modeling of SMT represents a useful tool for testing novel drugs and therapies. The successful replication of SMT native morphology demands scaffolds with an aligned anisotropic 3D architecture. In this work, a 3D PCL fibrous scaffold with aligned morphology was developed through the synergistic combination of Melt-Extrusion Additive Manufacturing (MEAM) and porogen leaching, utilizing PCL as the bulk material and PEG as the porogen. PCL/PEG blends with different polymer ratios (60/40, 50/50, 40/60) were produced and characterized through a DSC analysis. The MEAM process parameters and porogen leaching in bi-distilled water allowed for the development of a micrometric anisotropic fibrous structure with fiber diameters ranging from 10 to 100 μm, depending on PCL/PEG blend ratios. The fibrous scaffolds were coated with Gelatin type A to achieve a biomimetic coating for an in vitro cell culture and mechanically characterized via AFM. The 40/60 PCL/PEG scaffolds yielded the most homogeneous and smallest fibers and the greatest physiological stiffness. In vitro cell culture studies were performed by seeding C2C12 cells onto a selected scaffold, enabling their attachment, alignment, and myotube formation along the PCL fibers during a 14-day culture period. The resultant anisotropic scaffold morphology promoted SMT-like cell conformation, establishing a versatile platform for developing in vitro models of tissues with anisotropic morphology