Elien Gevaert

The 3D printing of gelatin methacrylamide cell-laden tissue-engineered constructs with high cell viability.

In the present study, we report on the combined efforts of material chemistry, engineering and biology as a systemic approach for the fabrication of high viability 3D printed macroporous gelatin methacrylamide constructs. First, we propose the use and optimization of VA-086 as a photo-initiator with enhanced biocompatibility compared to the conventional Irgacure 2959. Second, a parametric study on the printing of gelatins was performed in order to characterize and compare construct architectures. Hereby, the influence of the hydrogel building block concentration, the printing temperature, the printing pressure, the printing speed, and the cell density were analyzed in depth. As a result, scaffolds could be designed having a 100% interconnected pore network in the gelatin concentration range of 10-20 w/v%. In the last part, the fabrication of cell-laden scaffolds was studied, whereby the application for tissue engineering was tested by encapsulation of the hepatocarcinoma cell line (HepG2). Printing pressure and needle shape was revealed to impact the overall cell viability. Mechanically stable cell-laden gelatin methacrylamide scaffolds with high cell viability (>97%) could be printed.

An anti-inflammatory selective glucocorticoid receptor modulator preserves osteoblast differentiation

Glucocorticoids (GCs) are in widespread use to treat inflammatory bone diseases, such as rheumatoid arthritis (RA). Their anti‐inflammatory efficacy, however, is accompanied by deleterious effects on bone, leading to GC‐induced osteoporosis (GIO). These effects include up‐regulation of the receptor activator of NF‐κB ligand/osteoprotegerin (RANKL/OPG) ratio to promote bone‐resorbing osteoclasts and include inhibition of bone‐forming osteoblasts. We previously identified suppression of osteoblast differentiation by the monomer glucocorticoid receptor (GR) via the inhibition of Il11 expression as a crucial mechanism for GIO. Here we show that the GR‐modulating substance compound A (CpdA), which does not induce GR dimerization, still suppresses proinflammatory cytokines in fibroblast‐like synovial cells from patients with RA and in osteoblasts. In contrast to the full GR agonist dexamethasone, it does not unfavorably alter the RANKL/OPG ratio and does not affect Il11 expression and subsequent STAT3 phosphorylation in these cells. Notably, while dexamethasone inhibits osteoblast differentiation, CpdA does not affect osteoblast differentiation in vitro and in vivo. We describe here for the first time that selective GR modulators can act against inflammation, while not impairing osteoblast differentiation.—Rauch, A., Gossye, V., Bracke, D., Gevaert, E., Jacques, P., Van Beneden, K., Vandooren, B., Rauner, M., Hofbauer, L. C., Haegeman, G., Elewaut, D., Tuckermann, J. P., De Bosscher, K. An anti‐inflammatory selective glucocorticoid receptor modulator preserves osteoblast differentiation. FASEB J. 25, 1323–1332 (2011). www.fasebj.org

Quantitative contrasts in the photopolymerization of acrylamide and methacrylamide-functionalized gelatin hydrogel building blocks

The synthesis and evaluation of gelatin acrylamide as an alternative to the well-established gelatin methacrylamide are reported. High-resolution magic angle spinning NMR is used for the quantitative determination of the chemical cross-linking density. The gelatin acrylamide precursors reveal enhanced cross-linking in terms of reactivity and double bond conversion, resulting in stronger networks. Remarkably, even at very low double bond conversions, ≈5%, gel fractions of ≈40% are obtained. The cross-linked networks are also reviewed in the framework of the rubber elasticity and thermodynamic swelling theories to estimate important nanostructural properties. Preliminary cell tests revealed that highly viable (>90%) cell-laden constructs are obtained.

Barrier function of the nasal mucosa in health and type-2 biased airway diseases.

The mucosal lining of the upper airways represents the outer surface of the body to the ambient air and its contents and is prepared for it as the first line of defense. Apart from the well-described physical barrier and the mucociliary clearance, a variety of systems, including the airway microbiome, antimicrobial proteins, damage-associated molecular patterns, innate lymphoid cells, epithelial-derived cytokines and chemokines, and finally the adaptive immune system, as well as eosinophils as newly appreciated defense cells form different levels of protection against and response to any possible intruder. Of interest especially for allergic airway disease, mucosal germs might not just elicit a classical Th1/Th17-biased inflammatory response, but may directly induce a type-2 mucosal inflammation. Innovative therapeutic interventions may be possible at different levels also; however, whether modulations of the innate or adaptive immune responses will finally be more successful, and how the correction of the adaptive immune response might impact on the innate side, will be determined in the near future.

High Throughput Micro-Well Generation of Hepatocyte Micro-Aggregates for Tissue Engineering

The main challenge in hepatic tissue engineering is the fast dedifferentiation of primary hepatocytes in vitro. One successful approach to maintain hepatocyte phenotype on the longer term is the cultivation of cells as aggregates. This paper demonstrates the use of an agarose micro-well chip for the high throughput generation of hepatocyte aggregates, uniform in size. In our study we observed that aggregation of hepatocytes had a beneficial effect on the expression of certain hepatocyte specific markers. Moreover we observed that the beneficial effect was dependent on the aggregate dimensions, indicating that aggregate parameters should be carefully considered. In a second part of the study, the selected aggregates were immobilized by encapsulation in methacrylamide-modified gelatin. Phenotype evaluations revealed that a stable hepatocyte phenotype could be maintained during 21 days when encapsulated in the hydrogel. In conclusion we have demonstrated the beneficial use of micro-well chips for hepatocyte aggregation and the size-dependent effects on hepatocyte phenotype. We also pointed out that methacrylamide-modified gelatin is suitable for the encapsulation of these aggregates.

Viruses and bacteria in Th2 biased allergic airway disease

Allergic airway diseases are typically characterized by a type 2‐biased inflammation. Multiple distinct viruses and bacteria have been detected in the airways. Recently, it has been confirmed that the microbiome of allergic individuals differs from that of healthy subjects, showing a close relationship with the type 2 response in allergic airway disease. In this review, we summarize the recent findings on the prevalence of viruses and bacteria in type 2‐biased airway diseases and on the mechanisms employed by viruses and bacteria in propagating type 2 responses. The understanding of the microbial composition and postinfectious immune programming is critical for the reconstruction of the normal microflora and immune status in allergic airway diseases.

Advances in rhinitis and rhinosinusitis in 2015

The last year has seen great progress in the understanding of upper airway disease and in its management. For allergic rhinitis, authors focused on the prediction of and effect on the natural course of disease. New evidence was published for the disease-modifying effect of allergen immunotherapy in terms of avoidance of new sensitizations and prevention of asthma in either randomized or real-life studies. Specifically, for patients with house dust mite allergies, which are often underestimated and difficult to diagnose, the efficacy of SQ house dust mite sublingual immunotherapy tablets has been demonstrated in patients with allergic rhinitis and asthma. For the first time, allergen immunotherapy significantly reduced asthma exacerbations. In patients with chronic rhinosinusitis, a novel endotyping approach purely based on T helper cell biomarkers has been developed and has shown clinical relevance through associations with asthma comorbidity and recurrence after surgery. Severe nasal polyposis with high risk for asthma comorbidity and disease recurrence is characterized by type 2 inflammatory patterns, including IgE antibodies to staphylococcal superantigens; several studies using biologic agents have targeted exactly this spectrum of mediators. This goes in parallel with new knowledge on even more type 2 mediators derived from epithelial cells, which will expand the number of possible candidates for innovative intervention.

Extracellular eosinophilic traps in association with Staphylococcus aureus at the site of epithelial barrier defects in patients with severe airway inflammation

Background Chronic rhinosinusitis with nasal polyps is characterized by TH2‐biased eosinophilic inflammation. Eosinophils have been shown to generate so‐called extracellular eosinophilic traps (EETs) under similar pathologic conditions. Objective Our aim was to investigate a possible link between EET formation and the presence of Staphylococcus aureus, an organism frequently colonizing the upper airways, at the human mucosal site of the disease. Methods Tissue slides were investigated for the presence of EETs and S aureus by using immunofluorescent staining and the PNA‐Fish assay, respectively. An ex vivo human mucosal disease tissue model was used for artificial infection with S aureus. Cell markers were analyzed by using immunohistochemistry, the Luminex Multiplex assay, ELISA, PCR, and immunoblotting and linked to the presence of EETs. Results About 8.8% ± 4.8% of the infiltrating eosinophils exhibited EETs in patients’ nasal polyp tissues. Formation of EETs was associated with increased IL‐5 (P < .05) and periostin (P < .05) tissue levels and colonization with S aureus (P < .05). By using an ex vivo human mucosal disease tissue model, EET formation was induced (4.2 ± 0.9–fold) on exposure to S aureus but not Staphylococcus epidermidis. Eosinophils were shown to migrate (P < .01) toward S aureus and entrap the bacteria both inside and outside the mucosal tissue. Blocking NAPDH oxidase activity led to a complete inhibition (P < .05) of EET formation by S aureus. Conclusion Eosinophils are likely to be specifically recruited to S aureus and possibly other microorganisms and form EETs at sites of airway epithelial damage to protect the host from infections in patients with chronic rhinosinusitis with nasal polyps.

Galactose‐Functionalized Gelatin Hydrogels Improve the Functionality of Encapsulated Hepg2 Cells

The present study investigates the effect of galactosylated gelatin on encapsulated HepG2 cells. Methacrylamide modified gelatin is evaluated and compared with its galactosylated counterpart with respect to effects on viability, morphological characteristics, proliferation, and the expression of hepatocyte specific markers. The research reveals that further modifications of methacrylamide modified gelatin are possible without affecting the survival of the encapsulated cells (viability of 90%). Moreover, the study demonstrates a clear and long-term (up to 21 d) improvement in hepatocyte specific gene expression when the cells are encapsulated in the galactosylated gelatin. It is concluded that the use of galactosylated gelatin derivates supports the hepatocyte phenotype.

Staphylococcus aureus controls interleukin-5 release in upper airway inflammation

Staphylococcus aureus is a frequent colonizer of the upper airways in chronic rhinosinusitis with nasal polyps, but also resides intramucosally; it has been shown that secreted staphylococcal proteins such as enterotoxins and serine proteases induce the release of cytokines such as IL-5. We have analyzed nasal polyp tissue freshly obtained during routine surgery, which did or did not contain cultivatable S. aureus, to study spontaneous IL-5 production by nasal polyp tissue over 24 and 72h in tissue culture. In S. aureus-positive samples we interfered by killing the bacteria using antibiotics or S. aureus specific intravenous staphylococcal phages (ISP), active or heat-inactivated. Phage-neutralizing antibodies were used to demonstrate the specificity of the phage-mediated effects. We monitored S. aureus colony forming units, and identified S. aureus proteins by mass spectrometry. We demonstrate that cultivatable S. aureus may be found in type-2 inflamed nasal polyps; the pathogen is replicating within 24h and secretes proteins, including enterotoxins and serine proteases. The presence of S. aureus was associated with a significantly higher release of IL-5. Killing of S. aureus by antibiotics or specific ISP significantly reduced the IL-5 release. The suppressive activity of the bacteriophage on IL-5 be abolished by heat inactivation or anti-phage antibodies. BIOLOGICAL SIGNIFICANCE In this study, we used high resolution mass spectrometry to identify S. aureus proteins directly in infected nasal polyp tissue and nasal polyp tissue incubated over 24 and 72h in culture. We discovered bacterial proteins including enterotoxins and serine proteases like proteins. These experiments indicate a direct role of S. aureus in the regulation of IL-5 production in nasal polyps and may suggest the involvement of bacterial proteins detected in the tissues.