Lorenzo Sardelli

Thymic involution is commonly addressed as a loss of epithelial and lymphoid tissue, yet the accompanying remodelling of the microenvironment remains poorly defined. This work applied a biomaterials-centered approach to compare young and aged bovine thymus by integrating histology, oscillatory rheology, untargeted lipidomics, ICP-MS and AP-MALDI mass spectrometry imaging. This holistic approach connects the mechanical, compositional, and spatial features of the native thymus with the development of thymus-inspired biomaterials. Ageing increased both storage and loss moduli by more than one order of magnitude and reduced the linear viscoelastic region approximately fivefold, defining a markedly stiffer and more strain-sensitive material state. This mechanical transition was accompanied by lipid remodelling, with double realtive contribution of triacylglycerols to the lipid pool doubled and loss of membrane-associated phospholipids. The elemental profile also contracted, with total metal content decreasing by one-third and zinc showing a reduction of 70%. At the architectural level, the corticomedullary ratio was more than halved, while AP-MALDI imaging revealed an approximately 40% reduction in the annotated molecular repertoire and a shift from homogeneous to fragmented distributions of choline and representative phosphatidylcholine species. These results show that thymic ageing emerges from the concomitant variations of viscoelastic behaviour, chemical composition, and molecular organization. By defining these interconnected alterations, this study establishes a materials-based foundation for the design of bioinspired systems aimed at reproducing features of the young thymic niche and supporting future thymic repair.

P. aeruginosa employs specific quorum sensing (QS) mechanisms to orchestrate biofilm formation, enhancing resistance to host defences. In physiological conditions, QS molecules permeate the lung environment and cellular membrane to reach the cytoplasmic Aryl Hydrocarbon Receptor (AhR) that is pivotal for activating the immune response against infection. In pathological conditions like cystic fibrosis (CF) this interkingdom communication is altered, favouring P. aeruginosa persistence and chronic infection. Here, we aim to investigate the molecular journey of QS molecules from CF-like environments to the cytoplasm by quantifying via HPLC-MS the permeability of selected QS molecules (quinolones, lactones, and phenazines) through in vitro models of the two main biological lung barriers: CF-mucus and cellular membrane. While QS molecules not activating AhR exhibit intermediate permeability through the cellular membrane model (PAMPA) (1.0–4.0 × 10⁻⁶ cm/s), the AhR-activating molecule (pyocyanin) shows significantly higher permeability (8.6 ± 1.4 × 10⁻⁶ cm/s). Importantly, combining the CF mucus model with PAMPA induces a 50% decrease in pyocyanin permeability, indicating a strong mucus-shielding effect with pathological implications in infection eradication. This study underscores the importance of quantitatively describing the AhR-active bacterial molecules, even in vitro, to offer new perspectives for understanding P. aeruginosa virulence mechanisms and for proposing new antibacterial therapeutic approaches.

Bacterial‐derived molecules are at the basis of bacteria–bacteria and bacteria–host communication. In the context of cystic fibrosis (CF), they are considered possible therapeutic molecules for their natural binding capability on the immunomodulatory cytoplasmic aryl hydrocarbon receptor (AhR). An exponentially growing number of bacteria‐derived molecules are identified as AhR activators, highlighting the need for systems to screen possible lead candidates. This challenge is addressed by applying an in vitro tool mimicking the two main barriers that potential AhR‐targeting drugs must overcome: the cytoplasmic membrane and the CF pathological mucus. A small dataset of AhR ligands with potential therapeutic applications is selected. The apparent permeability of bacterial‐derived molecules across a cellular membrane model is quantified and molecules capable of reaching the cytoplasmic target (AhR) are identified. In a second step, a CF in vitro mucus model is integrated with the phospholipid membrane and the impact of mucus on permeability is assessed. Overall, this study proposes an integrated mucosal platform as a suitable tool in the emerging field of postbiotics as a therapeutic strategy for CF. The mucosal platform can enable the rapid identification of molecules compatible with cytoplasmic targeting of AhR among candidate‐drug representatives.

Hepatotoxicity‐related issues are poorly predicted during preclinical experimentation, as its relevance is limited by the inadequacy to screen all the non‐physiological subclasses of the population. These pitfalls can be solved by implementing complex in vitro models of hepatic physiology and pathologies in the preclinical phase. To produce these platforms, extrusion‐based bioprinting is focused on, since it allows to manufacture tridimensional cell‐laden constructs with controlled geometries, in a high‐throughput manner. Different bioinks, whose formulation is tailored to mimic the chemomechanical environment of hepatic steatosis, the most prevalent hepatic disorder worldwide, are proposed. Internally crosslinked alginate hydrogels are chosen as structural components of the inks. Their viscoelastic properties (G′ = 512–730 Pa and G″ = 94–276 Pa, depending on frequency) are tuned to mimic those of steatotic liver tissue. Porcine hepatic ECM is introduced as a relevant biochemical cue. Sodium oleate is added to recall the accumulation of lipids in the tissue. Downstream analyses on 14‐layered bioprinted structures cultured for 10 days reveal the establishment of steatotic‐like features (intracellular lipid vesicles, viability decrease up to ≈50%) without needing external conditionings. The presented bioinks are thus suitable to fabricate complex models of hepatic steatosis to be implemented in a high‐throughput experimental frame.

We developed an innovative millifluidic organ-on-a-chip device, named MINERVA 2.0, that is optically accessible and suitable to serial connection. In the present work, we evaluated MINERVA 2.0 as millifluidic gut epithelium-on-a-chip by using computational modeling and biological assessment. We also tested MINERVA 2.0 in a serially connected configuration prodromal to address the complexity of multiorgan interaction. Once cultured under perfusion in our device, human gut immortalized Caco-2 epithelial cells were able to survive at least up to 7 days and form a three-dimensional layer with detectable tight junctions (occludin and zonulin-1 positive). Functional layer development was supported by measurable trans-epithelial resistance and FITC-dextran permeability regulation, together with mucin-2 expression. The dynamic culturing led to a specific transcriptomic profile, assessed by RNASeq, with a total of 524 dysregulated transcripts (191 upregulated and 333 downregulated) between static and dynamic condition. Overall, the collected results suggest that our gut-on-a-chip millifluidic model displays key gut epithelium features and, thanks to its modular design, may be the basis to build a customizable multiorgan-on-a-chip platform.

The inflammatory bowels diseases (IBD) are autoimmune diseases that deeply impact the patients’ quality of life. The IBD pathogenesis is not yet defined, but evidence demonstrated that the IBD chronic inflammation is related to an impaired intestinal barrier. Traditionally, two actors were considered for their contribution to this disfunction: the gut microbiota and intestinal epithelium. However, a third element, which is the intestinal mucus, should be considered as peer of the epithelium and microbiota. Indeed, mucus represents the biological interface between bacteria and cells, filtering molecules or toxins and preventing bacteria penetration exploiting both structural and compositional properties. The boosting effect of the mucus characterization towards IBD comprehension is far too underestimated, although some mucus-oriented studies are already reported in literature. This work reviews the intestinal barrier features, describing each component of the gut mucosa (i.e., epithelium, microbiota, and mucus) in a mucus-oriented perspective.